The Mechanism of Autooxidation of Myoglobin* · 2001-06-21 · toward the CD corner. iments for determining the mechanism of autooxidation are presented under "Results." MATERIALS

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Soeiety for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 10, Issue of April 5, pp. 6995-7010, 1993 Printed in U. S. A .

The Mechanism of Autooxidation of Myoglobin* (Received for publication, August 24, 1992)

Robert E. Brantley, Jr.$$, Stephen J. Smerdonll, Anthony J. Wilkinsonllll, Eileen W. Singleton$, and John S. Olson$** From the $Department of Biochemistry and Cell Biology and the W. M. Keck Center for Computational Biology, Rice University, Houston, Teras 77251 and the TDepartment of Chemistry, University of York, Heslington, York YO1 5 0 0 , United Kingdom

Time courses for the autooxidation of native and mutant sperm whale and pig myoglobins were measured at 37 "C in the presence of catalase and superoxide dismutase. In sperm whale myoglobin, Hise4(E7) was replaced with Gln, Gly, Ala, Val, Thr, Leu, and Phe; Valss(Ell) was replaced with Ala, Ile, Leu, and Phe; Leu2'(B10) was replaced with Ala, Val, and Phe. In pig myoglobin, His'"(E7) was replaced with Val; Vale8(E11) was replaced with Thr and Ser; Thr"(El0) was replaced with Ala, Val, Glu, and Arg; Lys4'(CD3) was replaced with Ser, Glu, His, and Arg. The observed pseudo-first order rate constants varied over 4 orders of magnitude, from 58 h" (H64A) to 0.055 h" (native) to 0.005 h" (L29F) at 37 "C, pH 7, in air. The dependences of the observed autooxidation rate constant on oxygen concentration and pH were measured for native and selected mutant myoglobins.

In the native proteins and in most mutants still pos- sessing the distal histidine, autooxidation occurs through a combination of two mechanisms. At high [O,] , direct dissociation of the neutral superoxide radical (HOn) from oxymyoglobin dominates, and this process is accelerated by decreasing pH. At low [O,], autooxidation occurs by a bimolecular reaction between molecular oxygen and deoxymyoglobin containing a weakly coordinated water molecule. The neutral side chain of the distal histidine (Hise4) inhibits autooxidation by hydrogen bonding to bound oxygen, pre- venting both H02 dissociation and the oxidative bimolecular reaction with deoxymyoglobin. Replacement of His64 by amino acids incapable of hydrogen bonding to the bound ligand markedly increases the rate of autooxidation and causes the superoxide mechanism to predominate. Increasing the polarity of the distal pocket by substitution of Vale8 with Ser and Thr accel- erates autooxidation, presumably by facilitating protonation of the Fe(I1). O2 complex. Increasing the net anionic charge at the protein surface in the vicinity of the heme group also enhances the rate of autooxidation. Decreasing the volume of the distal pocket by replacing small amino acids with larger aliphatic or aromatic residues at positions 68 ( E l 1) and 29 (B10) inhibits autooxidation markedly by decreasing the accessibility of the iron atom to solvent water molecules.

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

of Health Training Grant GM-08280. § Recipient of a graduate fellowship from the National Institutes

11 Supported by Grant GR/E 98867 from the Science and Engi- neering Research Council, UK.

** Supported by National Institutes of Health Grants GM-35649 and HL-47020, Grant C-612 from the Robert A. Welch Foundation, and the W. M. Keck Foundation.

In addition to regulating oxygen affinity, the protein structure of myoglobin and hemoglobin serves to keep the heme complex in the reduced (Fe(I1)) state. This is a crucial function since the oxidized (Fe(II1)) state is incapable of binding oxygen and thus physiologically inactive. Free heme in solution autooxidizes very rapidly, whereas native myoglobin autooxidizes in hours. Oxidation of the heme iron atom is also the first step in the denaturation of these proteins since globin has a much lower affinity for ferric hemin than the reduced form. In both myoglobin and hemoglobin, hemin loss at 37 "C eventually leads to unfolding and precipitation. In order to reverse autooxidation and inhibit denaturation, enzymatic reduction systems are present in red cells and muscle tissue to keep these proteins in the reduced state (Bunn and Forget, 1986).

The first studies to examine the dependence of the rate of autooxidation on pH and oxygen concentration were carried out in 1935 by Brooks for ox hemoglobin and in 1952 by George and Stratmann for bovine myoglobin. The data of George and Stratmann (1952) are shown in Fig. 1, A and E , where k,. represents the pseudo-first order rate constant for autooxidation. As shown, this rate constant exhibits a bell- shaped dependence on [O,] which is centered around the equilibrium dissociation constant ( K d ) for oxygen binding. At high [O,], k,, approaches an asymptote which is not equal to zero. Empirical equations were given that described the observed oxygen dependence, but no mechanistic interpretations were offered.

Since this early work, several chemical mechanisms have been suggested to explain the autooxidation of hemeproteins. Weiss (1964) proposed that an electron is transferred from the reduced iron to the bound oxygen causing direct dissociation into oxidized protein and free superoxide radical:

Mb(Fe(II))02 - Mb+(Fe(III)) + 0; (Eq. 1)

where kdisa is the first order rate constant for superoxide dissociation. Evidence in support of this mechanism was presented by Misra and Fridovich (1972) who showed that epinephrine is co-oxidized to adrenochrome in solutions of oxyhemoglobin and that a significant portion of the co-oxidation of epinephrine is inhibited by superoxide dismutase. Wallace et al. (1982) showed that cytochrome c is reduced at a rate equivalent to that for the autooxidation of oxymyoglobin (or oxyhemoglobin) in mixtures of the two proteins. Although indirect, this evidence suggests strongly that the superoxide radical is the initial reduced product of autooxidation. Wallace et al. (1982) and Shikama and Sugawara (1978) also showed that the rate of autooxidation increases markedly with decreasing pH. Shikama (1984) proposed that protonation of the Fe(I1) '0, complex is required to allow dissociation of neutral HO, from the active site of oxymyoglo-

kdw

6995

6996 Mechanism of Autooxidation of Myoglobin

h

L c . 3 v

X

A

0.8 ax> A

George and Stratmann (1952) Native Bovine Myoglobin

30C, pH 5.7

0.4 \, bimolecular

0.2 "I " -"- """"

unimolecular

0 1 0 20 3 0

dissolved oxygen (pM)

"-1 0.6

Native Bovine Myoglobin

30C, pH 5.7 George and Stratmann (1952)

B I

0.0 0 2 0 0 400 600 800 1000 1200


FIG. 1. Effect of oxygen concentration on the rate of autooxidation of 200 BM native bovine myoglobin at 30 "C in 600 mM potassium phosphate, pH 5.7 (taken from George and Stratmann, 1962). Calculated dependences for the unimolecular and bimolecular mechanisms were combined to fit the observed data.

bin since it seems likely that the anion would immediately re- associate with ferric iron.

The simple dissociative mechanism predicts that the rate of autooxidation should depend on the fractional saturation of the reduced hemeprotein, Y, and that for myoglobin the rate constant can be represented by:

where Kd is the equilibrium Oz dissociation constant. This expression predicts that k,. should increase with increasing [02] and asymptotically approach a limiting value a t high oxygen tensions. However, as shown in Fig. L4, k,, exhibits a bell-shaped dependence on [02], and thus, the unimolecular mechanism by itself is not sufficient to explain autooxidation.

Wallace et al. (1982), Satoh and Shikama (1981), and others have shown that the rate of autooxidation is enhanced by anionic ferric ligands. Shikama proposed a reductive displacement reaction in which the nucleophile entered the distal pocket of oxymyoglobin and directly facilitated superoxide dissociation (Shikama, 1984). As pointed out by Wallace et

al. (1982), this concerted model cannot account for the 2- to %fold decrease in k,. with increasing [02] which was observed by George and Stratmann (1952, Fig. 1). Wallace et al. (1982) proposed a ligand-mediated electron transfer mechanism in which nucleophiles induce electron transfer from reduced heme to free oxygen in a bimolecular reaction. Two steps are required for this mechanism. First, the facilitating nucleophile binds to unliganded ferrous myoglobin. This binding must be slow and weak since there is no spectral evidence for direct coordination of azide or water to the iron atom in deoxymyoglobin. The second, oxidative step involves the reaction of Oz with the 6-coordinate ferrous heme complex in an outer sphere mechanism. The weakly bound nucleophile facilitates this reaction by acting as a strong ligand for the ferric state. These reactions may be represented by the following equations:

MbOz + Mb + 0 2 Kd

(Eq. 3)

kl MbN + 0 2 2 Mb'N + 0; 0%. 5)

where Kd is the equilibrium dissociation constant; kl and k-l are the association and dissociation rate constants, respectively, for the binding of the nucleophile, N, to deoxymyoglobin; and kz is the bimolecular rate constant for the reaction of Oz with the 6-coordinate ferrous complex.

A single, pseudo-first order rate constant can be derived for this reaction scheme by assuming 0, binding is very rapid and assuming a steady-state and very low level of the transient intermediate MbN (see Wallace et al., 1982 and Appendix B):

Unlike unimolecular superoxide dissociation, this bimolecular mechanism can account for the bell-shaped portion of the oxygen dependence reported by George and Stratmann (Fig. 1A). At low [OZ], the rate will be directly proportional to [OZ], whereas the rate will be inversely proportional to [O,] at high [O,]. However, this mechanism predicts that k,. should approach zero at very high oxygen levels which is in conflict with the experimental observations in Fig. 1.

Utilizing native and mutant sperm whale and pig myoglobins constructed previously for studies on ligand binding, we have shown that the overall autooxidation rate dependence on oxygen and pH can be accounted for by a combination of the mechanisms and ideas presented above. In sperm whale myoglobin, Hisw was replaced with Gln, Gly, Ala, Val (also in pig myoglobin), Thr, Leu, and Phe to examine the effects of polarity and size of the distal residue. In sperm whale myoglobin, Valrn was replaced with Ala, Ile, Leu, and Phe, and Leuz9 was replaced with Ala, Val, and Phe to examine the effects of distal pocket size. In pig myoglobin, Val6' was replaced with Ser and Thr to examine more conservatively the effects of distal pocket polarity. In pig myoglobin, T h P was replaced with Ala, Val, Glu, and Arg and Lys45 was replaced with Ser, Glu, His, and Arg to examine the effects of residues on the heme periphery. The locations of these residues are shown in Fig. 2 which presents the distal pocket structures of oxy- and deoxymyoglobin (Phillips, 1980 and 1981; Phillips and Schoenborn, 1981). Control experiments describing the effects of catalase and superoxide dismutase, the effects of different methods of reduction of ferric samples, and comparisons between wild-type and native proteins are presented under "Materials and Methods." Derivations of the rate equations are presented in the Appendix. The key exper-

Mechanism of Autooxidation of Myoglobin 6997

A ? ?

RRG 115

I

\

d% HIS 93

FIG. 2. Distal pockets of the oxy ( A ) and deoxy ( B ) complexes of sperm whale myoglobin. The coordinates for panels A and B were taken from the structures of MbOp (Phillips, 1980) and Mb (Phillips, 1981; Phillips and Schoenborn, 1981). The ORTEP drawing in panel B was generated from a back view, looking from Ilelo7 across the heme group out toward the solvent; the drawing in panel B was generated from a side view, looking through the E helix, toward the CD corner.

iments for determining the mechanism of autooxidation are presented under "Results."

MATERIALS AND METHODS

Source of Proteins-Native sperm whale myoglobin in the oxidized or ferric form was purchased from Sigma prior to the ban on whale products. Further purification with ion exchange high performance liquid chromatography had no effect on the kinetic, equilibrium, and autooxidation rate constants measured for this protein. All plasmids for single HisM and Valrn mutants of sperm whale myoglobin, except AlaM, ThrM, and Leuss, were constructed by Barry Springer and Karen Egeberg at the University of Illinois, Urbana as described previously (Springer et al., 1989; Egeberg et al., 1990). Mutant sperm whale myoglobins containing L29A, L29V, and L29F replacements were constructed as described by Carver et al. (1992). Those mutants containing H64A, H64T, and V68L were constructed at Rice following the procedures of Egeberg et al. (1990). Wild-type and mutant sperm whale myoglobins were expressed and purified as described previously (Springer and Sligar, 1987; Carver et al., 1992). Native pig heart myoglobin was purified as described by Wittenberg and Wittenberg (1981). Wild-type and pig mutant myoglobins were expressed as fusion proteins and purified as described by Smerdon et al. (1991). Protein purity was assessed by either SDS-polyacrylamide gel electrophoresis or the ratio of the absorbance of the Soret peak (409 nm

for oxidized protein and 418 nm for reduced protein) to the absorbance at 280 nm. Proteins judged to be greater than 99% pure by SDS- polyacrylamide gel electrophoresis gave ratios of about 3 to 4.

Measurement of Autooxidation Rate Constants-Recombinant sperm whale myoglobins were expressed as holoproteins in Esche- richia coli, and, if sufficiently stable, were isolated in the reduced oxygenated state. The autooxidation rates of these proteins (wild- type, H64Q, V68A, V68L, V681, V68F, L29A, L29V, and L29F) were measured by simply incubating the sample at 37 "C in a cuvette a t the appropriate pH and [O,]. Typically, 60 pl of 1 mM oxymyoglobin was diluted directly into about 3 ml of 100 mM potassium phosphate a t pH 7 containing 1 mM EDTA and 3 mmol/mol of heme catalase and superoxide dismutase. Oxidation of the sample was followed either by recording entire visible spectra or by recording the decrease in absorbance a t a single wavelength, usually 581 nm, in a Shimadzu 2101 UV-Vis spectrophotometer equipped with a six-cell changer and thermoelectric temperature control device (CPS-260). Almost all the autooxidation reactions were characterized by only two spectral species with sharp isosbestic points, and the observed time courses could be represented by a simple first order process under each set of conditions, d(MbO,)/dt = -k,,(MbO,), where kx is the apparent autooxidation rate constant. Unless the rate was extremely slow or precipitation became significant, reactions were followed to near completion to ensure that the observed time course was monophasic. In some cases, endpoint absorbance values were obtained by adding a 10% excess of potassium ferricyanide to oxidize all the heme groups. The absorbance traces were fitted to a single exponential expression with an offset using an iterative, nonlinear least squares algorithm. The offset represented the absorbance of the metmyoglobin product at the wavelength of observation and was either allowed to vary or input as a fixed value based on ferricyanide oxidation.

Native pig heart myoglobin was isolated in the reduced oxy form. All recombinant pig myoglobins, stable or unstable, were isolated in the oxidized form and had to be reduced chemically. Native sperm whale myoglobin purchased from Sigma and unstable mutants (H64G, H64A, H64V, and H64T) were also obtained in the oxidized state and had to be reduced prior to reaction with oxygen. H64L and H64F sperm whale myoglobin have markedly high affinities for carbon monoxide and are expressed and isolated from Escherichia coli in the reduced state containing bound CO. This endogenously bound ligand had to be removed by oxidation with K3Fe(CN)6, and the resultant ferric sample was gel-filtered to remove excess ferricyanide. These proteins were then re-reduced and reacted with oxygen.

Those mutant myoglobins which exhibit markedly high autooxidation rates (>2 h-I) had to be reduced anaerobically in tonometers and then reacted with oxygen in a rapid-mixing device. These experiments were performed in a Gibson-Dionex stopped-flow apparatus equipped with an On-Line-Instruments-Systems (OLIS) model 3820 data collection system. The flow system was made oxygen-free by prolonged incubation with concentrated solutions of deoxyhemoglo- bin. Protein was mixed with buffer equilibrated with 1 atm of high purity mixtures of Np and O2 or with air, and the absorbance change of the mixture was followed a t either 406, 420, or 581 nm.

Effects of Catalase and Superoxide Dismutase on the Rate of Au- tooxidation-Measurements of hemeprotein autooxidation are com- plicated by side reactions with the oxidation products HpOp, OT, and HO and by the presence of trace metals and other molecules which can mediate electron transfer (Rifkin, 1974; Shikama, 1984; Watkins et al., 1985; Wazawa et al., 1992). Side reactions with radicals may generate covalent adducts with the heme and between proteins (Tew and Ortiz de Montellano, 1988 Catalan0 et al., 1989). When myoglobins are isolated in their oxidized form, they must be reduced, usually with sodium dithionite, and the products of this reaction may also enhance the autooxidation rate (Antonini and Brunori, 1971). In view of these previous results, we were obliged to test the different reduction techniques and eliminate the problems associated with side reactions before beginning intensive mechanistic and mutagenesis studies.

The effects of catalase and superoxide dismutase on the autooxidation rate of sperm whale native myoglobin were studied a t several oxygen concentrations. Both enzymes were varied from 0 to 30 mmol per mol of heme (0 to 9.4 X 10" units of catalase, Sigma-S2515, and 0 to 265 X lo7 units of superoxide dismutase, Sigma-C40, per mol of heme) for the autooxidation of native sperm whale myoglobin at 37 "C, pH 7, in 0.5% Op-equilibrated buffer (data not shown). The maximal reduction in autooxidation rate was obtained a t 3 mmol per mol of heme, a value very close to the optimum concentration used by Watkins et al. (1985) for similar studies with hemoglobin. This


optimum is roughly 12-fold greater than that found for the ratio of catalase and superoxide dismutase to heme in the red cell (Carrel1 et al., 1978). As shown in Fig. 3, even with both enzymes at 3 mmol per mol of heme, less than a 30% reduction in the rate of autooxidation was observed at 37 "C, pH 7, regardless of oxygen concentration. Thus, the idea of Wazawa et al. (1992) that the bell-shaped dependence of k,. on [Oz] is an artifact of H202 production appears to be incorrect. Even though the reduction in the rate of autooxidation was small, all reactions were carried out in the presence of the optimal amount of catalase and superoxide dismutase (3 mmol/mol of heme).

Effect of Reduction Procedure on the Rate of Autooxidation-Three reduction protocols were used to generate oxymyoglobin from ferric samples: 1) chemical reduction with excess dithionite followed by gel filtration; 2) anaerobic, stoichiometric reduction with dithionite; and 3) photochemical reduction. In the first method, 20-100 pl of -1 mM ferric myoglobin was injected into a small Microfuge tube containing a few grains of sodium dithionite using a gas-tight Hamilton syringe equipped with a fixed needle. Immediately after injection and disso- lution of the dithionite (1-2 s), the solution was drawn back into the syringe, loaded into the upper layer of a small (1 to 3 ml bed volume) G-25-medium Sephadex gel filtration column, and eluted as quickly as possible (see Rohlfs et al. (1990) and Brantley (1992)).

In the second method, ferric samples were loaded into a glass tonometer at concentrations ranging from 5-20 pM and then made anaerobic by several cycles of vacuum degassing and nitrogen flushing. While under a considerable positive pressure of nitrogen, a ground glass fitting was removed from one end of the tonometer and replaced with a Hamilton syringe, custom made with its own ground glass fitting. The syringe contained a concentrated, anaerobic solution of sodium dithionite, and the protein was reduced stoichiometrically by adding small aliquots of the reducing agent. Reduction was followed by tilting the solution into a sidearm equipped with a cuvette and monitoring the spectrum in the visible range.

In the third, photochemical reduction method, two protocols were tried. Initially, the procedure described by Springer et al. (1989) was used. Native sperm whale protein was loaded into a tonometer containing 100 mM potassium phosphate, pH 7,25 mM EDTA and made anaerobic by several cycles of vacuum degassing and nitrogen flushing. The solution was illuminated by a xenon arc lamp for about an hour, and reduction was followed spectrophotometrically. When reduction was complete, the tonometer was opened and shaken in air to obtain the oxycomplex. Alternatively, illumination in the presence of 5-deazaflavin was used to accelerate the rate of reduction. In this case, the sample buffer contained only 5 mM EDTA 5-deazaflavin in

Native Sperm 37C, pH 7.0

Whale Myoglobin

0 0 mmol CatalaselSOD per mol heme A 3 mmol Catalase/SOD per mol heme

0.0 0 200 400 600 800 1000

dissolved oxygen (uM)

FIG. 3. Effect of catalase and superoxide dismutase on the rate of autooxidation of 20 p~ native sperm whale myoglobin as a function of oxygen concentration at 37 "C, pH 7, in 100 mM potassium phosphate, 1 mM EDTA. Error bars represent the standard deviation from the mean with N 2 3.

methanol was added to 0.2 PM; and catalase and superoxide dismutase were present at 3 mmol per mol of heme. The tonometer was made anaerobic in the dark with cycles of vacuum degassing and nitrogen flushing, and then the solution was illuminated on ice under several flood lamps for 1 h. Again, when reduction was complete, the tonometer was opened, and the solution was shaken in air to obtain the oxycomplex.

A comparison of autooxidation rates for a series of different oxymyoglobin samples and reduction protocols is shown in Table I. A detailed discussion of these controls has been presented by Brantley (1992), and three basic conclusions apply. First, chemical reduction with dithionite does not affect the rate of autooxidation if only stoichiometric amounts are added or if excess reducing agent and its oxidation products are removed quickly by gel filtration. The autooxidation rate constants obtained with and without dithionite reduction for sperm whale wild-type A, wild-type B, V68A, V681, and V68F myoglobins and for native pig myoglobin are indistinguishable, considering the &10-30% error in $.. Oxy samples treated with dithionite and then gel-filtered or oxidized with ferricyanide and then re-reduced with dithionite show autooxidation rates similar to those of the untreated samples (Table I, column 3). As a futher verification of the chemical reduction procedure, two experiments were carried out with native sperm whale myoglobin using conditions similar to those reported previously. In air-equilibrated phosphate buffer at pH 7, 25 "C, we observed a rate constant of 0.012 h" for native sperm whale myoglobin, which is equivalent to the value of 0.011 h" reported by Shikama and Sugarawa (1978) for bovine myoglobin under the same conditions. Wallace et al. (1982) reported a bimolecular rate constant of 3.84 M" h" for bovine myoglobin at pH 5.7, 30 "C, in air and the presence of 13 mM azide; under the same conditions, we observed a rate constant of 3.25 M" h" for native sperm whale myoglobin.

The second conclusion is that photochemical reduction results in higher rates of autooxidation. For example, the rate constant reported for native and wild-type myoglobin photochemically reduced in the presence of high EDTA (0.26-0.36 h-') is roughly 6 times greater than that obtained using the dithionite reduction, gel filtration procedure (0.055 h-'). The cause of this artifactual increase is unknown. Rate constants for samples obtained using deazaflavin-mediated photoreduction were also systematically greater than those for proteins reduced with the dithionite procedure (Table I, column 7). In this case, deazaflavin probably acts as an electron mediator to speed up the rate of autooxidation.

The third conclusion is that the autooxidation rates of native myoglobins and wild-type proteins expressed in E. coli are very similar. The original sperm whale wild-type myoglobin expressed in E. coli is denoted wild-type A and contains an additional methionine at the N terminus. As pointed out by Phillips et al. (1990), the first gene was constructed using the originally published, but incorrect Asn at position 122 (Edmunson, 1965) instead of Asp (Romero- Herrara and Lehmann, 1974). X-ray crystallography has shown wild- type A to be identical in tertiary structure with native sperm whale myoglobin except for the N-terminal region (Phillips et al., 1990). Subsequently, Dr. Barry Springer incorporated the correct codon for Asp'22 into the synthetic gene, generating wild-type B. Previous studies have shown wild-type A to be identical with native sperm whale myoglobin by various spectroscopies and ligand binding kinetics (Rohlfs et al., 1990). Furthermore, native, wild-type A and wild- type B myoglobins show identical kinetic and thermodynamic constants for 02, CO, and azide binding.

Measurement of the Autooxidation Rate as a Function of Oxygen Concentration-For these experiments, oxymyoglobin solutions were placed in tonometers equipped with a 1-cm path length, side arm cuvette and equilibrated with the appropriate gas mixture of oxygen and nitrogen. Those samples which were obtained in the ferric form and which were relatively stable to autooxidation were reduced with dithionite aerobically and gel-filtered before being placed in the tonometers. Mutants with very high autooxidation rates were titrated anaerobically with dithionite in tonometers which could be attached directly to a Gibson-Dionex stopped-flow apparatus. These solutions were then rapidly mixed with varying concentrations of oxygen. A t low 02/heme ratios, the [O,] varies as the autooxidation reaction proceeds, increasing for those myoglobins with high oxygen affinities and decreasing for those proteins with low affinities. This complica- tion is discussed in detail in Appendix A.

Measurement of the Autooxidation Rate as a Function of pH-For simple spectrophotometric measurements on time scales of 1 or more hours, the proteins were diluted into the appropriate air-equilibrated buffer: pH 3, potassium phosphate; pH 4, potassium citrate; pH 5,


TABLE I Effect of reduction procedure on the autooxidation rate constant for selected myoglobins at 37 "C, p H 7.0

Values displayed in the ferrous columns were obtained for samples purified as oxymyoglobin, whereas those in the ferric columns were obtained with protein purified as metmyoglobin. No DT means no dithionite was used. DT, air denotes dithionite reduction in the presence of air. DT, N2 denotes anaerobic, stoichiometric dithionite reduction. Deazaflavin denotes anaerobic photoreduction in the presence of 5- deazaflavin. EDTA-photo denotes values reported by Barry Springer (1989) using photoreduction in the presence of 25 mM EDTA. The asterisk denotes the value measured by us for native sperm whale myoglobin simulating the conditions described by Springer (1989). The error values represent the standard deviation from the mean for replicate samples with N 2 3.

b. (h")

Protein Ferrous samples Ferric samples

No DT DT, air DT, air DT, N2 Deazaflavin EDTA-photo

Pig native Pig wild-type SW native Wild-type A Wild-type B H64Q H64G H64V H64L H64F V68A V68I V68F

0.051 f 0.005 0.074 f 0.001 0.058 f 0.003 0.070 f 0.006 0.054 f 0.006

0.045 f 0.003 0.05 f 0.005 0.057 f 0.003 0.082 f 0.001 0.094 f 0.008 0.074 f 0.006 0.18 f 0.02 0.24 f 0.02

31 f 9 35 f 9

9 9 f 3

0.26 f 0.01 0.32 f 0.04 0.75 f 0.02 0.811 f 0.005

0.067 f 0.001 0.071 f 0.008 0.068 f 0.008

potassium acetate; pH 6, potassium maleate; pH 7, potassium phosphate; pH 8, potassium phosphate, glycylglycine, or Tris phosphate; pH 9, sodium bicarbonate or bis-Tris propane' phosphate. The pH values of all solutions were adjusted at 37 "C. For stopped-flow experiments, the proteins were reduced anaerobically in tonometers using either 10 mM potassium phosphate, 1 mM EDTA, pH 7 or 15 mM Tris phosphate, 1 mM EDTA, pH 8.5 as a buffer. These solutions were then rapidly mixed with the appropriate concentrated buffer to achieve the desired final pH. Control mixing experiments were done without protein to determine what the pH of the concentrated buffer solution had to be in order to yield the desired pH of the reaction mixture. The ionic strength of all final assay solutions was 0.22 to 0.25 M. The exact conditions are given by Brantley (1992). The pH of solutions which were incubated for a long time was measured after the reaction to ensure that no changes had occurred. All buffers contained 1 mM EDTA, and all final solutions contained catalase and superoxide dismutase, each at 3 mmol/mol of heme.

Measurement of the Oxygen Dissociation Equilibrium Constant, Kd-Kd values for all proteins were determined at 37 "c by measuring the kinetics of 0 2 displacement by CO (see Olson (1981)):

When a solution of an oxymyoglobin complex (MbO,) is mixed rapidly with a solution of CO under pseudo-first order conditions (02,CO >> Mb), the observed replacement rate, robs, is given by:

When [O,] is much greater than [ C O ] , the equation may be simplified and rob is given by:

If the rate constants k'co and kc0 are known, Kd can be calculated by rearranging this expression to:

(Eq. 10)

Those samples which were isolated as MbO, were used directly. All the metmyoglobin samples were reduced with ditbionite and gel-

' The abbreviation used is: bis-Tris propane, 1,3-bis[tris(hydroxymethyl)methylamino]propane.

0.089 f 0.002 0.105 f 0.022 0.049 f 0.002 0.093 f 0.022

0.095 f 0.008 0.172 f 0.028

44 33 f 3

10 6

0.069 f 0.001 0.12 f 0.01

0.36 (0.23*) 0.35

0.34 39 36

16 0.62 1.23 0.27

filtered on a Sephadex G-25-medium column that had been washed thoroughly with buffer equilibrated with the appropriate mixture of 0, and N,. The eluted protein was collected directly into a septum- stoppered glass syringe containing phosphate buffer and the appropriate concentration of 0,. The oxymyoglobin sample was then quickly mixed with a CO solution in the stopped-flow apparatus to determine rob.

The value of k'co at 37 "C was measured by diluting the protein in anaerobic buffer containing a few grains of dithionite to ensure reduction to deoxymyoglobin. This sample was then reacted with various concentrations of CO under pseudo-first order conditions at 37 "C, again in the stopped-flow apparatus. The value of kco at 37 "C was determined by measuring the rate of CO displacement from the corresponding carbon monoxide complexes using an excess of NO (Olson, 1981). The resultant values of robs, k'co, and kc0 were then used to compute the Kd for 0, binding at 37 "C as described above.

RESULTS

Effect of Oxygen on the Rate of Autooxidation of Native Myoglobin-Wallace and Caughey (1979) have reported that the rate of autooxidation of hemoglobin in the presence of anions does not approach zero in oxygen-free solutions, im- plying the presence of some incidental oxidizing agent in the reaction mixture. However, when wild-type' sperm whale myoglobin was prepared anaerobically in a tonometer with cycles of vacuum degassing and nitrogen flushing, little or no oxidation was observed. There was a very small, initial decrease in absorbance at 581 nm, but then no further change occurred for over 3 days (Fig. 4). This result is consistent with a very small amount of oxygen being present in the initial solution. Then, after consumption of this small amount of oxygen, the reaction stopped due to the lack of an oxidizing reagent. The same result was obtained in the presence of 30 mM azide (Fig. 4). Thus, oxygen can be assumed to be the sole oxidizing agent, and the rate of autooxidation of myoglobin a t zero oxygen concentration is zero.

As shown in Fig. 5, our results for the dependence of k,, on [02] can be simulated assuming that autooxidation results from a combination of the unimolecular superoxide dissociation mechanism (Equation 1) and the bimolecular mechanism

Wild-type sperm whale myoglobin in the main text refers to wild- type A myoglobin which has an Asn residue at position 122 instead of Asp as in the native protein (see "Materials and Methods").


." 0 2 0 4 0 6 0 80

time (hours)

FIG. 4. Autooxidation of 20 PM wild-type sperm whale myoglobin at 37 "C, in air- or nitrogen-equilibrated 100 mM potassium phosphate, l mM EDTA, pH 7, in the presence (squares) or absence (circles) of 30 mM sodium azide. Catalase and superoxide dismutase were each present at 3 mmol/mol of heme.

(Equations 3-5). Since the data were measured in the absence of added anionic ferric ligands, the facilitating nucleophile (N) is assumed to be water. When both mechanisms apply and the steady-state level of MbN is very small (see Appendix B), ko, is given by:

The limiting value at high oxygen levels is the rate of superoxide dissociation, kdiss. If the & for oxygen binding is known, the rate of unimolecular HOz dissociation can be subtracted leaving the contribution from the bimolecular mechanism. The resultant values for kbimolecu~ar show a bell-shaped curve centered around the K d for O2 binding and become zero at high oxygen levels. Rearrangement of Equation 11 shows that k 1 and k2 cannot be fitted for individually and must be determined as a ratio. Since the maximum value for kox is often found at [02] f K d , the derivative of Equation 11 shows that k-l/kz must also n&. The rate of nucleophile binding to the ferrous iron atom can then be estimated a t high oxygen levels as kbimolecular approaches zero, and kl[N] has a value of -1.1 h" for wild-type sperm whale myoglobin at 37 "C, pH 7 , in the absence of nucleophiles other than water.

The solid line drawn through the data points of George and Stratmann in Fig. lA was also computed using Equation 11. Thus, the complex oxygen dependence of the autooxidation of native myoglobin (and presumably hemoglobin) can be explained by a combination of the superoxide dissociation reaction of Weiss (1964) and the bimolecular mechanism of Wallace et al. (1982). The contributions of the two mechanisms at different oxygen levels are shown in Fig. 5 for our results with native sperm whale myoglobin. Rates of autooxidation at oxygen levels lower than -5 PM were not measured since, in this range, large standard deviations are obtained and the free concentration of oxygen changes markedly as the reaction proceeds (see Appendix A).

Effect of Anions-Wallace et al. (1982), Satoh and Shikama (1981), and others have shown that the rate of autooxidation

c . d Y

X

Y

- L c . v 3

X

0.3 R A

37C, pH 7.0 Native Sperm Whale Myoglobin

bimolecular

."."""---

---------"- """"" V ~ """I

0 200 400 600 800 1000


B

Native Sperm Whale Myoglobin 37C, pH 7.0

bimolecular *\.

unimolecular

*. -9 -"

-99 * 9- --- --",


FIG. 5. Effect of oxygen concentration on the rate of autooxidation of 20 FM native sperm whale myoglobin at 37 "C, in 100 mM potassium phosphate, 1 mM EDTA, pH 7, containing 3 mmol/mol each of heme catalase and superoxide dismutase. Calculated dependences for the unimolecular and bimolecular mechanisms were combined to simulate the observed data. Error bars represent the standard deviation from the mean with N 2 3. The value at zero oxygen is a genuine data point.

is enhanced by anionic ferric ligands. The dependence of k,, on [ 0 2 ] as a function of azide concentration is shown in Fig. 6A for native sperm whale myoglobin at pH 7 , 37 "C. At 1 atm of oxygen, k,, is independent of azide concentration, but as the oxygen concentration is lowered to 6 PM, the rate of autooxidation is significantly enhanced by this anion. This result strongly supports our view that the mechanism of autooxidation of native myoglobin is a combination of the superoxide dissociation and bimolecular mechanisms. At high [02], the predominant mechanism is superoxide dissociation which should be unaffected by azide concentration. At low


Sperm Whale Myoglobin 37C, pH 7.0

1.2

1.0

0.8

0.6

0.4

0.2

0.0 0 200 400 600 800 1000

0 OmMazide 0 30 m M azide 0 100 mM azide


Sperm Whale Myoglobin 37C, p H 7.0

0 0 m M chloride 0 150 m M chloride

B I

V."

0 200 400 600 800 1000


FIG. 6. Effect of azide (A) and chloride ( B ) concentration on the rate of autooxidation as a function of oxygen concentration for 20 PM native sperm whale myoglobin at 37 "C, in 100 mM potassium phosphate, 1 mM EDTA, pH 7, containing 3 mmol/mol each of heme catalase and superoxide dismutase. Error bars represent the standard deviation from the mean with N 2 3. In panel A , the lines drawn through the points are not theoretical because of the complexity of adding a second bimolecular term to Equation 11.

[02], the ligand-enhanced, bimolecular mechanism dominates and by definition should increase in rate with increasing nucleophile concentration (see Appendix B). Wallace et al. (1974,1982) reported a dependence of autooxidation on chloride concentration using bovine myoglobin and human hemoglobin, whereas Satoh and Shikama (1981) reported no effect of chloride on the autooxidation rate of bovine myoglobin. Our results agree with Satoh and Shikama and show that the rate of autooxidation of native sperm whale myoglobin at pH 7, 37 "C is unaffected by physiological concentrations of chloride, at all oxygen levels (Fig. 6B). Thus, the facilitating nucleophile for the bimolecular mechanism under physiological conditions is probably water.

Correlation of Autooridation Rate with the Kd for O2 Bind- ing-Previous studies on the autooxidation of native and mutant sperm whale myoglobins (Springer et al., 1989) have shown a proportionality between the oxygen equilibrium dissociation constant and the rate of autooxidation in air-equilibrated solutions a t 20-37 "C. This inverse relationship be-

tween O2 affinity and k,, is readily explained for the bimolecular mechanism since at high [02], Equation 6 reduces to kbimoleeular = kl [N] K d / [ 0 2 ] . However, the contribution of the bimolecular mechanism to the observed rate diminishes at high [O,], and the unimolecular dissociation of superoxide becomes the dominant process. According to Equation 11, the rate of superoxide dissociation should be equal to kdiss and independent of both Kd and [02] as long as the protein is fully saturated with ligand. Thus, proportionality between Kd and k,, is not necessarily required at high [02] if both mechanisms are operating simultaneously.

However, we have confirmed experimentally that in general there is a direct correlation between K d and k,, using geneti- cally engineered pig and sperm whale myoglobins. A plot of log kox uersus log Kd is shown in Fig. 7 for 27 different myoglobins. Even with the large scatter in the data, proportionality is generally observed and this correlation suggests strongly that the rate of superoxide dissociation is regulated by the same structural features that govern the equilibrium dissociation constant for O2 binding.

Effects of Distal Pocket Mutations-Sample time courses for wild-type and mutant sperm whale and pig myoglobins are shown in Fig. 8 and show a 10,000-fold range of rate constants. Most of the slowly autooxidizing mutants and the native and wild-type proteins exhibit monophasic time courses. Fits to time courses for the less stable mutants, particularly the H64G and H64V proteins at all oxygen concentrations and H64Q and V68A myoglobins a t low oxygen concentrations, show small systematic deviations from a single exponential expression. Better fits were obtained using a two-exponential expression; however, the rate constant for the faster phase was only a factor of 2 or 3 greater than that for the slower phase, and the amplitude of the faster phase was always -50-80% of the total absorbance change. The improvement in the fit was not great enough to justify more extensive analysis, and the rates presented in Tables I, 11, and

- 3 . 2 1 0 1 2

log kox

FIG. 7. Plot of log of the autooxidation rate constant (k0J uersu8 log of the oxygen equilibrium dissociation constant (Ks) for 27 different native and mutant sperm whale and pig myoglobins at 37 "C, in 100 mM potassium phosphate, 1 mM EDTA, pH 7, containing 3 mmol/mol each of heme catalase and superoxide dismutase.


0 2 4 6 8 10

time (hours)

FIG. 8. Normalized absorbance time courses for the autooxidation of selected sperm whale and pig myoglobins at 37 OC in air-equilibrated 100 mM potassium phosphate, 1 mM EDTA, pH 7, containing 3 mmol/mol each of heme catalase and superoxide dismutase.

TABLE I1 Autooridation rate and oxygen equilibrium dissociation constants

for position 64 (E7) mutants of sperm whale and pig myoglobins i n air at 37 "C, p H 7.0

The error values represent the standard deviation from the mean with N 2 3. The means for native, wild-type, and H64Q proteins were computed as the average of the values obtained from different reduction experiments excluding the EDTA and photoreduction methods (see Table I). For the other H64 proteins, the error values represent the standard deviation from the mean of three or more kinetic time courses with two or more independently prepared samples. Y,, indicates the fractional saturation of myoglobin with oxygen in air- equilibrated buffer a t 37 "C.

t. Kd,O? yair

h" P M

Sperm whale myoglobins H64G 44 f 9 46 0.84 H64A 58 f 11 110 0.70 H64V 33 f 9 200 0.56 H64T 54 f 5 190 0.58 H64L 10 100 0.72 H64F 6 340 0.43 H64Q 0.21 f 0.03 15 0.94 H64 (native) 0.055 f 0.005 4.0 0.98 H64 (E. coli) 0.051 f 0.006 3.6 0.98

H64V 25 250 0.51 H64 (native) 0.061 f 0.012 5.0 0.98 H64 (E. coli) 0.07 f 0.01 4.1 0.98

Pig myoglobins

I11 for these mutants were taken from the fits to a single exponential expression. These parameters do have large standard deviations and should be viewed with some caution.

Autooxidation rate constants and Kd values for oxygen binding to native, wild-type, and HisM mutants of sperm whale and pig myoglobin are shown in Table 11. Native and wild- type myoglobins exhibit identical rate constants, and k,, for the H64Q mutant was increased only 4-fold from 0.055 h" to 0.21 h-' in air at pH 7, 37 "C. As noted by Springer et al. (1989), substitution with apolar residues at position 64 causes

100-800-fold increases in K,, compared to wild-type myoglobin. The rate constants for the apolar H64G, H64A, H64V, H64L, and H64F mutants are 40, 60, 30, 10, and 6 h-', respectively, a t 37 "C, pH 7, in air-equilibrated buffer, and, thus, the rate of autooxidation does depend on the size of the apolar side chain at position 64.

The dependence of the autooxidation rate for the H64Q mutant on oxygen is shown in Fig. 9A. The form of the dependence is similar to that for native sperm whale myoglobin with a roughly 4-fold increase in the rate over the whole range of oxygen tensions. As shown in Fig. 9B, the observed rate for the autooxidation of the sperm whale H64V mutant shows a hyperbolic dependence on [02] unlike the more com- plicated bell-shaped dependence shown by native myoglobin in Figs. 1 and 4. This result suggests strongly that autooxidation of the H64V mutant occurs only by the superoxide dissociation pathway. The concentration of oxygen which gives one-half the maximal value of k,, is identical with the K d value measured for O2 binding as predicted by Equation 2. Clearly, the H64V substitution is facilitating HOz dissociation. Putting Thr at position 64 produces a small additional increase in the rate of autooxidation (Table 11) but little effect

1.0 A

0.8 Sperm Whale Myoglobin 37C, pH 7.0

0 Native 0 H64Q

0.6

0.4

0.2

n 1, u.u 0 200 4 0 0 6 0 0 no0 1000


50 B

40 -

H64V Sperm Whale Myoglobin 37C, pH 7.0

0 200 4 0 0 6 0 0 800 1000 "

0 200 4 0 0 6 0 0 800 1000


FIG. 9. Effect of oxygen concentration on the rate of autooxidation of 20 p~ native and H64Q ( A ) and 10 p~ H64V ( B ) sperm whale myoglobins at 37 OC, in 100 mM potassium phosphate, 1 mM EDTA, pH 7, containing 3 mmol/mol each of heme catalase and superoxide dismutase. Error bars represent the standard deviation from the mean with N 2 3. The two circles at 260 pM oxygen in panel A represent two different experiments.


on oxygen affinity compared to the H64V mutant. This increase in ko. for the ThrM mutant is probably due to an increase in polarity of the distal pocket compared to that in the VaP4 mutant; however, the effect is quite small and just outside the error limits.

Autooxidation rate constants and oxygen equilibrium dissociation constants for wild-type and Valrn mutants of sperm whale and pig myoglobin are shown in Table 111. The rate constants for sperm whale myoglobins containing Ala, Val (native), Leu, Ile, and Phe at position 68 are 0.26, 0.051, 0.10, 0.75, and 0.069 h-', respectively. Thus, the effects of changing the size of residue 68 on the autooxidation rate constant are complex and not straightforward to interpret. The oxygen concentration dependences of k,, for sperm whale native, V68A, and V68F myoglobins a t 37 "C, pH 7, are shown in Fig. 1OA. The results indicate that the major effect of the V68A mutation is an increase in kdss for the unimolecular mechanism since the autooxidation rate for all three proteins con- verge a t low oxygen concentrations.

As shown in Table 111, the V68S and V68T substitutions cause 20- and 50-fold increases, respectively, in the rate of autooxidation of pig myoglobin. These results demonstrate unequivocally that increasing the polarity of the distal pocket causes marked increases in the rate of autooxidation. The [O,] dependence for the autooxidation of V68T indicates that the superoxide dissociation pathway has become dominant and that the dramatic increase in k,, is due to a large increase in kdss (Fig. lOB).

The effects of mutation at position 29 are shown in Table IV and were presented previously by Carver et al. (1992). As the size of residue 29 is increased from Ala to Val to Leu, there is a 4-fold decrease in k,,, even though the K d for oxygen binding is relatively unaffected. Substitution of leucine with phenylalanine produces a dramatic 16-fold decrease in the K d for oxygen binding and a 10-fold decrease in the rate of autooxidation. The half-life for the L29F mutant is -6 days a t 37 "C, pH 7, compared to a half-life of 13 h for wild-type myoglobin.

Autooxidation rate constants and 0, equilibrium dissociation constants for wild-type pig myoglobin, position 45, and position 67 mutants are given in Table V. The values of k,, for wild-type, K45R, K45H, K45S, and K45E were 0.07, 0.10, 0.17, 0.40, and 1.20 h-', respectively. Although the O2 equilibrium dissociation constant for the K45R substitution decreased slightly compared to wild-type myoglobin, the O2 equilibrium dissociation constants for the other position 45 mutants were increased roughly 2-fold. The K45E substitu-

TABLE 111 Autooridation rate and oxygen equilibrium dissociation constants for position 68 (El I ) mutants of sperm whale and pig myoglobins

in air at 37 "C, p H 7.0 The error values represent the standard deviation from the mean

with N 5 3. Yair indicates the fractional saturation of myoglobin with oxygen in air-equilibrated buffer at 37 "C.

Sperm whale myoglobins V68A V68 (E. coli) V68L V68I V68F

V68S V68T V68 (E. coli)

Pig myoglobins

h"

0.26 f 0.01 0.051 f 0.006

0.10 0.75 f 0.02 0.069 f 0.002

1.4 f 0.01 3.5 f 0.3 0.07 f 0.01

4.0 0.98 3.6 0.99

16 3.5 0.99

0.94 5.8 0.98

50 5.0 0.98

0.84 5.0 0.98

L c . 3 v

X

A

0.4 A

Sperm Whale Myoglobin 37C, pH 7.0

0.3

0.2

0.0 ' I 0 200 400 600 800 1000


B

.I

- L c . z 2

A V68T

Pig Myoglobin 37C, pH 7.0

x

0 Wild-type

1t

0 I 0 200 400 600 800 1000


FIG. 10. Effect of oxygen concentration on the rate of autooxidation of 20 p~ native, V68A, and V68F sperm whale myoglobins ( A ) and 20 p~ wild-type and V68T pig myoglobins ( E ) at 37 "C, in 100 mM potassium phosphate, 1 mM EDTA, pH 7, containing 3 mmol/mol each of heme catalase and superoxide dismutase. Error bars represent the standard deviation from the mean with N 2 3.

TABLE IV Autooridation rate and oxygen equilibrium dissociation constants for position 29 (BlO) mutants of sperm whale myoglobin in air at 37 "C,

p H 7.0 The error values represent the standard deviation from the mean

with N 2 3. Yair indicates the fractional saturation of myoglobin with oxveen in air-eauilibrated buffer at 37 "C.

Sperm whale myoglobins k,. Id.0, YaLr

h" P M

L29A 0.24 f 0.1 5.0 0.98 L29V 0.23 f 0.05 4.6 0.98 L29 (E. coli) 0.051 k 0.006 3.6 0.99 L29F 0.005 0.22 0.999

tion replaces a positive with a negative charge, enhances the rate of autooxidation -17-f01d, and causes the superoxide dissociation pathway to become dominant as judged by the dependence of k,. on [O,] (Fig. 11). Intermediate increases were exhibited by the K45S and K45H mutants, whereas the


TABLE V Autooxidation rate and oxygen equilibrium dissociation constants

for position 45 (CD3) and 67 (ElO) mutants of pig myoglobin in air at 37 "C, p H 7.0

The error values represent the standard deviation from the mean with N 2 3. Yer indicates the fractional saturation of myoglobin with oxygen in air-equilibrated buffer at 37 "C.

Pig myoglobins k o x K.0, Y, h" B M

Wild-type (E. coli) 0.07 f 0.01 4.0 0.98 K45S 0.4 8.4 0.97 K45E 1.2 f 0.1 K45H

8.6 0.97 0.17

K45R 6.0 0.98

0.10 f 0.01 T67A

2.8 0.99 0.9

T67V 2.0 0.99

0.1 T67E

3.8 0.98 0.2

T67R 2.0 0.99

0.037 f 0.009 3.0 0.99

X

3

0.5 I A K45E 0 Wild-type

"." 0 200 400 600 800 1000

dissolved oxygen @M)

FIG. 11. Effect of oxygen concentration on the rate of autooxidation of 20 IM wild-type and K46E pig myoglobins at 37 "C, in 100 mM potassium phosphate, 1 mM EDTA, pH 7, containing 3 mmol/mol each of heme catalase and superoxide dismutase. Error bars represent the standard deviation from the mean with N 2 3.

rate constant for K45R myoglobin was increased less than 2- fold. The values of k,. for wild-type, T67R, T67V, T67E, and T67A myoglobins were 0.07, 0.04, 0.10, 0.20, and 0.90 h-', respectively. Thus, placing a positive charge at position 67 decreases the rate of autooxidation 2-fold, adding a negative charge increases k,. -3-fold, and decreasing the size from Thr to Ala causes a 10-fold increase.

Effect of p H on the Autooxidation Rate Constunt-The marked increase in the autooxidation rate of hemeproteins with decreasing pH was noted as early as 1935 and has been confirmed by several groups (Brooks, 1935; George and Strat- mann, 1952; Shikama and Sugawara, 1978; Wallace et al., 1982). Shikama (1984) and others have suggested that the distal histidine is a part of a proton-relay system that is necessary for the autooxidation reaction and that protonation of this residue speeds up superoxide dissociation. In order to test this proton-relay mechanism more rigorously, we examined the pH dependence of autooxidation for native and

several mutant sperm whale and pig myoglobins. As shown in Fig. 12A, mutation of the distal histidine causes

dramatic increases in the absolute values of the autooxidation rate constants with the trend H64V >>> H64Q > native, regardless of pH. However, when the values are normalized to pH 7, the relative effects of pH are roughly the same for all the proteins studied (Fig. 12B). Thus, a proton relay mechanism involving the distal histidine is not necessary since equivalent pH dependence was observed for the H64V mutant which has no polar group in the vicinity of the Fe(I1).

" I A

2 4 6 8 10

PH

I

-" Pig native B * K45E

"t V68T -0- sw native

H64Q * H64V

\

2 4 6 8

PH

10

FIG. 12. Effect of pH on the rate of autooxidation for several native and mutant sperm whale and pig myoglobins at 37 "C in air-equilibrated 100 mM potassium phosphate, 1 mM EDTA, pH 7, containing 3 mmol/mol each of heme catalase and superoxide dismutase. The log of the absolute rate constant is plotted against pH in panel A. In panel B, the log of the ratio of the rate constant at a given pH to the rate constant at pH 7 is plotted against pH. Error bars represent the standard deviation from the mean with N 2 3. For the H64V mutant (pH 5-9) and the K45E and V68T mutants (pH < 7), the error bars represent the standard deviation from the mean of three or more kinetic time courses using one or two prepared samples. The H64Q mutant precipitates rapidly at pH 5 6.


O2 complex. Thus, the increase in autooxidation rate with decreasing pH for these proteins is more readily interpreted as due to the equilibrium protonation of the Fe(I1). 0 2 complex to form Fe(II).02H+ which then rapidly dissociates into Fe(II1) and H02.

It is unlikely that the increase in k,, in going from pH 9 to 7 is due to an increase in the bimolecular contribution to the observed autooxidation rate. First, the Kd for oxygen binding and, indeed all ferrous ligand binding rate parameters, are independent of pH in this range. Second, autooxidation of both K45E and H64V myoglobin proceeds only through the unimolecular pathway with no apparent contribution from the bimolecular pathway (Figs. 9B and l l) , and the unimolecular reaction is the dominant pathway in air-equilibrated buffer at 37 "C, pH 7, for the autooxidation of native and V68T myoglobins. Third, if water is the nucleophile that enhances the bimolecular contribution to k,,, the rate constant for its binding to the reduced iron, kl in Equations 12- 14, is probably independent of pH as well. If hydroxide were the nucleophile, k,. should have increased with increasing pH. Thus, the pH dependence observed in the physiological range is most likely due to an increase in the rate of unimolecular superoxide dissociation, in Equation 11. Since the pH dependence is relatively independent of the structures of the distal pocket amino acids (Fig. 12B), it seems likely that the protonated form of the iron-02 complex, (Fe(I1). 02H+), is the dissociating species.

DISCUSSION

Chemical Mechanism of Autooxidation-In order to corre- late the observed data for both native and mutant myoglobins, we propose the chemical mechanism for autooxidation given in Scheme I. The complex dependence of k,, on oxygen concentration for native myoglobin suggests two different

H-His H-His

-Fe(ll)- -Fe(III) I I

H-His,,, 'H H

-Fe(lll)- v I

(Mb+H20)

pathways for autooxidation. The starting species is the Fe(I1). O2 complex with a hydrogen bond between the bound oxygen and the N, atom of the distal histidine. The next steps involve disruption of the hydrogen bond and then either dissociation into Mb and O2 or protonation of the Fe(I1). O2 complex. In the former case, autooxidation involves a bimolecular reaction between free O2 and Mb whereas in the latter case autooxidation occurs as a direct result of HO2 dissociation. Both cases result in the production of aquometmyoglobin and superoxide radicals.

The oxygen, pH, and nucleophile dependence of autooxidation and the correlation between K d and k,, are consistent with this chemical mechanism. The bell-shaped oxygen dependence of the autooxidation rate constant requires a bimolecular pathway involving the reaction between free O2 and a weakly bound nucleophile-deoxymyoglobin complex. At high [O,] where little deoxymyoglobin is present, the limiting, non-zero rate constant suggests strongly that a unimolecular superoxide dissociation pathway occurs. The autooxidation rate is independent of azide concentration a t high [02] where superoxide dissociation dominates but becomes increasingly dependent on azide at low [02] where the bimolecular mechanism is the major pathway for autooxidation. The increase in autooxidation rate with decreasing pH in the range from 9 to 7 occurs with or without a distal histidine supporting the idea that the Fe(I1) .02 complex must be protonated before superoxide can dissociate. Interpretation of the results in the pH range 7-5 is more difficult. Below pH 6, both the proximal and distal histidines become protonated, altering markedly the rate and equilibrium constants for ligand binding and presumably autooxidation (Coletta et al., 1985; Sage et al., 1991). In addition, the rate of hemin dissociation and protein denaturation increases markedly under acidic conditions (Bunn and Forget, 1986).

Role of His64 and Hydrogen Bonding-Scheme I provides a simple explanation for the increase in k,, with apolar substitutions for the distal histidine. Loss of the hydrogen bond between residue 64 and the bound oxygen increases the total fraction of MbOz molecules that are capable of autooxidizing by either the bimolecular or unimolecular pathway. This prediction can be analyzed quantitatively in terms of the fractions of species present in the oxycomplex as shown in Scheme 11. Species B is the inert Fe(II).02 complex with a hydrogen bond to residue 64. Species C is the simple Fe(I1). 0 2 complex in which the partial negative charge on the oxygen atom is not neutralized by surrounding protein residues. Spe- cies A is the acidified Fe(II).02H+ complex which rapidly dissociates into Fe(II1) and HOz. The fractions ( f ) of these species present are determined by the strength of the hydrogen bond with residue 64 ( K B = [B]/[C]) and the acid dissociation constant for protonation of the Fe(I1). O2 complex ( K A

= [H+] [C]/[A]). Exact expressions for fA, fB, and fc are given in Scheme I1 (and see Appendix B). Again, the observed autooxidation rate constant, k,,, is determined by contribu-

His-H

2 Kg

H-His H-His

-Fd(Il)- 8 , KA f + I

, -Fe(Il)_ pFe(lI)p I 7

(MQ) ( M b W H+ (Mb02H+)

B C A

I

fg= K A K B fc= K A K A ( K B + ~ ) + ( H + )

( H + ) KA(KB+I)+(H+) f A = KA(KB+I)+(H+)

SCHEME I SCHEME I1

7006 Mechanism of Autooridation of Myoglobin

tions from the unimolecular and bimolecular mechanisms which are defined by:

kox = 4imoleeular + kunimoleeular (Eq. 12)

where K'd is the equilibrium dissociation constant for O2 release from species C in Scheme I1 and kldiss is the rate constant for superoxide dissociation from species A. Thus, the overall Kd and k&ss parameters from Equation 11 can be interpreted in terms of the microscopic parameters and the fractions of the C and A species, respectively:

Kd = K'dfc = K'd&

KA(KB + 1) + ( H + ) ' (Ea. 15)

When His@ is replaced with an apolar residue, K B = 0 and both fA and fc must increase significantly. As a result, the rate of autooxidation by both mechanisms will increase markedly at high [ 0 2 ] , since under these conditions, ~ n i m o l e e u l a r =: k'disa f~ and kt,imo~eeu~er = kl [N] K'd fc/[OZ]. This effect accounts for the correlation between ko, and Kd even when the pathway for autooxidation is exclusively superoxide dissociation. In- creasing the strength of hydrogen bonding decreases the overall Kd by -~ /KB and the fraction of species C and A by roughly the same amount (Scheme 11). At low [02], loss of the hydrogen bond between His64 and bound O2 should cause little effect since k,, =: kl [N] k2 [02]/k-, and does not depend directly on either fc or fA. However, mutation of residue 64 (and 29) may affect kl, k l , and k2.

The smallest increase in k,. was observed for the H64Q mutant, presumably because the amide side chain can still form an hydrogen bond with the bound 02, although KB is definitely smaller. The oxygen concentration dependence of k,. for the H64Q mutant shown in Fig. 9A suggests that the rates for both autooxidation pathways are increased by roughly the same amount. The 3- to 4-fold increases in k,, and Kd are readily interpreted to be due to a 3- to 4-fold decrease in KB.

Interpretation of the general trend of decreasing k,, with increasing size of the apolar residue at position 64 is somewhat equivocal. For the Gly64, Ala64, Valw, Phe@ series there is little correlation between ko, and Kd (Table 11). The O2 concentration dependence of the H64V mutant suggests that the unimolecular dissociation pathway predominates (Fig. 9B). Since the experiments described in Table I1 were carried out in air and the Kd values for the mutants are high, none of the mutants was saturated with oxygen. As a result, the ko. value will be less than k&,,. However, even when the ~ n i m o l e e u l a r

expression of Equations 12-14 is used to correct for the low values of fractional saturation (Y in Table 11), there is still a 5-fold difference between the autooxidation rates (kaSs) for the H64G and H64V mutants (-50 h-') compared to those for the H64L and H64F mutants (-10 h-'). The cause of this decrease is probably an increase in the acid dissociation constant for the Fe(I1). 02H+ complex when it is masked from solvent water by large apolar groups at position 64. As shown in Scheme 11, a larger value of KA would decrease the fraction of Fe(I1). 02H+ and hence the unimolecular rate constant.

Role of Va168, Steric Hindrance, and Distal Pocket Polarity- The V68S and V68T mutants were originally constructed by

Smerdon et al. (1991) to examine the effects of added distal pocket polarity on ligand binding. Azide binding parameters and the crystal structure of the mutant protein suggested that the water bound to Fe(II1) is stabilized considerably by interactions with T h P . However, the oxygen affinity of the mutant is decreased 17-fold. The crystal structure of the aquomet form of V68T pig myoglobin showed that the P-OH of the threonine side chain is within hydrogen bonding distance of both the main chain carbonyl group of the distal histidine and the oxygen atom of the bound water. The P-OH of the threonine side chain donates a proton to the main chain carbonyl oxygen of residue 64. As a result, the nonbonding electron pairs of the P-OH oxygen atom are pointed toward the ligand bound to the iron atom. In aquometmyoglobin, the bound water can form a hydrogen bond to the ThP8 side chain by donating a hydrogen atom to the hydroxyl oxygen atom. In oxymyoglobin, the Thr68 side chain destabilizes the bound 0 2 either by electrostatic repulsion of the nonbonding elec- trons on the adjacent oxygen atoms or by competing for the N, proton of the His64 side chain (see Smerdon et al., 1991).

The increase in the autooxidation rate for the V68T mutant is readily explained by these structural observations. Weak- ening the hydrogen bond between bound O2 and His64 would decrease KB and increase the fraction of reactive species. The proximity of the negative portion of the P-hydroxyl would destabilize bound O2 in general and increase Kd. This latter interaction would also stabilize the Fe(I1). 02H+ complex, perhaps by direct hydrogen bonding, causing a decrease in K A . The @-OH of the V68T mutant could also stabilize H 2 0 binding to ferrous deoxymyoglobin and enhance the rate of bimolecular autooxidation. However, the oxygen concentration dependence shown in Fig. 10B suggests that the superoxide dissociation pathway predominates. Presumably, the same arguments apply to the V68S mutant, the major difference being the greater flexibility of the hydroxymethyl side chain.

The simplest interpretation of the increase in k,, for the V68A mutant is the proposal by Egeberg et al. (1990) that the naturally occurring Val residue indirectly stabilizes bound O2 by orienting the ligand for more efficient hydrogen bonding to His64. Steric considerations indicate that this residue itself actually hinders oxygen binding by restricting the rotational freedom of the bound O2 and distorting the Fe-0-0 bonds. The latter effects serve to explain why the V68A substitution increases the affinity of myoglobin for CO roughly 2- to 3- fold, whereas a combination of less hindrance but weaker hydrogen bonding accounts for the lack of change in 0 2

affinity. In terms of Schemes I and 11, the V68A replacement would decrease both KB and K'd. The decrease in KB would increase the fraction of reactive C and A species, whereas the K d change would move the maximum value for ko, to a lower [O,]. The increase in volume of the distal pocket due to the V68A substitution could also increase the accessibility of the iron atom to water and other solvent nucleophiles. An increase in solvent accessibility could decrease KA, or increase the kl

[N] term in Equations 12-14, both of which would enhance the net rate of autooxidation. The results in Fig. 1OA suggest that the former effect dominates since ko, for the V68A mutant is &fold larger at high [02] but equal to that for the native protein at low [ 0 2 1 .

The V68F substitution produces very little change in the rate of autooxidation at all oxygen concentrations. There appears to be a compensation between increasing the hydro- phobicity of the distal pocket by the phenyl group which would inhibit autooxidation and weakening the H i ~ ~ ~ - 0 2 hydrogen bond due to the lack of a substituted P-carbon. The


6 -

5 -

4 -

3 -

2 -

1 -

Pig Heart Myoglobin, 20C

+ K45E * Native

Pig Heart Myoglobin, 20C

+ K45E * Native

Y

3 4 5 6 I

PH FIG. 13. Effect of pH on the CO association rate constants

of native and K45E pig myoglobins at 20 OC, in 100 mM potassium phosphate, 1 mM EDTA, pH 7.

situation for the V68I mutation is even less clear. The increase in Kd for oxygen binding is clearly due to steric hindrance and evidently this effect enhances autooxidation by increasing k'diss in Equations 12-14.

Role of Leuz9 and the Effect of Distal Pocket Size-The 4- fold increase in k,, when Leuz9 is replaced with Val or Ala can be explained by an increase in the size of the distal pocket and, hence, its accessibility to solvent. The dramatic 15-fold increase in oxygen affinity for the L29F mutant has been ascribed to a direct interaction between the partially positive edge of the phenyl ring and the partial negative charge on the second atom of the bound oxygen (Carver et al., 1992). This increased electrostatic stabilization of bound oxygen would be expressed as an increase in KB and a decrease in K d in Schemes I and 11. As in the case of hydrogen bonding to His64, an increase in KB would decrease both the bimolecular and unimolecular autooxidation rates. In addition, the phenylalanine substitution should decrease markedly the accessibility of the bound Oz to solvent water by filling the cavity directly adjacent to it.

Effects of Charge and Polarity at Positions 45 and 67 on the Heme Periphery-The electrostatic interactions involving

(Arg45 in sperm whale), Asp", His64, surface water molecules, and the heme-6-propionate shown in Fig. 2B are thought to stabilize the "closed door" position of the distal histidine and enhance its ability to donate a hydrogen bond to bound oxygen. Replacement of Lys45 with Arg should strengthen these interactions and does increase the affinity of pig myoglobin for oxygen %fold, by either enhancing the hydrogen bonding potential of the neutral form of His64 or adjusting the iron-heme geometry (Carver et al., 1991). The rate of autooxidation of the K45R mutant was increased, but the effect was very small (only 1.7-fold higher), and this mutation can be considered conservative with regard to autooxidation. The crystal structure of K45R metmyoglobin determined by Oldfield et aL3 is similar to that of the wild- type protein with Arg4' taking up a conformation akin to that of Arg4' in sperm whale myoglobin (Fig. 2). The K45H sub-

Oldfield, T. J., Smerdon, S. J., Dauter, Z., Petratos, K., Wilson, K. S. and Wilkinson, A. J. (1992) Biochemistry 31,8732-8739.

stitution results in a %fold increase in the rate of autooxidation. Depending on the pK, of the imidazole group in this environment, His45 could interact with Asp", His64, and the heme-6-propionate, although not as strongly as lysine or arginine.

Replacement of Lys45 with the smaller, uncharged serine residue causes a large 7-fold increase in the rate of autooxidation. In the K45S crystal structure, the exposed face of the heme is clearly more open, indicating increased accessibility of the distal pocket to solvent nucleophile^.^ In addition, the loss of positive charge at position 45 should enhance protonation of the bound Oz by reducing unfavorable electrostatic interactions between residue 45 and the Fe(I1) .OzH' complex. Both of these effects would facilitate autooxidation.

The K45E substitution causes a dramatic 17-fold increase in the rate of autooxidation. Introduction of the negative charge at residue 45 is likely to disrupt the favorable electrostatic interactions found in this region of the native protein, and this disruption is expected to increase the accessibility of the distal pocket to solvent and solute molecules. However, only small increases in the rates of Oz and CO binding to this pig myoglobin mutant were observed by Carver et al. (1991). Thus, rather than an increase in accessibility, the principal cause for the increase in the autooxidation rate of K45E pig myoglobin is probably the introduction of an anionic group near bound oxygen. This negative charge should facilitate protonation of either His64 or the Fe(I1). Oz complex. Evidence for the former effect has been obtained by comparing the pH dependence of CO binding to the reduced deoxy form of the K45E mutant and native pig myoglobins.

As shown in Fig. 13, the rate of CO binding increases markedly at pH values below 6 for native pig myoglobin. Part of this increase in the CO association rate constant has been attributed to protonation of the distal histidine (Quillin et al., 1992; Ormos et al., 1988; Morikis et al., 1989; Zhu et al., 1992). When His64 is protonated, it swings out of the distal pocket opening a channel from the solvent to the iron atom, enhancing the rate of ligand binding (Quillin et al., 1992). A similar increase in k'co is observed for the K45E mutant, but at significantly higher pH values (Fig. 13). Since much smaller or no increases in k'co were observed at low pH for mutants without His64 (Quillin et al., 1992), the enhanced pH effect for the K45E substitution is most readily interpreted as an increase in the pKa of the distal histidine. If the protonated side chain of His64 stays in the distal pocket and hydrogen bonds to bound Oz, the resultant complex is a tautomeric form of species A in our autooxidation mechanism (Scheme 11). Thus, protonation of the position 64 imidazole should facilitate the formation of Fe(I1). OzH+ and increase knimolecular markedly. The dependence of k,, on [O,] for the K45E substitution suggests strongly that enhanced protonation of bound Oz is the major cause of the increase in k,, since the reaction appears to proceed solely by the unimolecular pathway (Fig. 11).

The lack of crystal structures for the Thr'j7 mutants prevents unambiguous interpretations of the changes in autooxidation rates produced by these substitutions. VaP7 is found naturally in dog myoglobin, and this isosteric T67V substitution causes only small changes in k,, and Kd. Replacement of threonine with alanine may increase the accessibility of the distal pocket to water molecules and partially disrupt the hydrogen bonding lattice involving T h P , Lys45 (Arg4' in sperm whale), Asp", Hisa, and the heme-6-propionate. Either process could account for the increase in the rate of autooxidation caused by the T67A mutation.

The 2-fold decrease in k,, for T67R pig myoglobin is most


easily explained by electrostatic considerations. Introduction of another positively charged residue near the heme pocket should further inhibit protonation of the bound oxygen and its subsequent dissociation as neutral superoxide. This interpretation is supported by the increase in k,, for the T67E mutation. By analogy with the results for the K45E mutation, introduction of a negatively charged residue near the heme pocket at position 67 also creates an environment favoring protonation of Fe(I1). O2 and an enhanced rate of superoxide dissociation.

CONCLUSIONS

The data in Figs. lA, 4, and 5A show that both the unimolecular H 0 2 dissociation mechanism proposed by Weiss (1964) and the bimolecular reaction mechanism proposed by Wallace et al. (1982) occur during the autooxidation of native myoglobin and that the relative importance of each pathway depends on the absolute oxygen concentration (Scheme I). In air- equilibrated buffer at 37 "C, the dominant mechanism is direct, unimolecular superoxide dissociation for native myoglobin and all of the mutants examined.

The hydrogen bond provided by the neutral imidazole side chain of Hisa plays the most crucial role in the inhibition of autooxidation of myoglobin. This interaction prevents both dissociation of bound oxygen and its protonation. Replace- ment of His64 with apolar residues causes 100- to 800-fold increases in the rate of autooxidation because of the loss of this hydrogen bonding interaction. In addition, the dependence of k,, on [O,] for these rapidly oxidizing mutants indicates that the superoxide dissociation pathway is dominant under all conditions. The same relative pH dependence of k,, was observed for native and selected mutant proteins includ- ing some with substitutions at the distal histidine position. This result suggests that protonation of the Fe(11). O2 complex accounts for most of the pH dependence observed at or above p H 7.0. Further evidence in favor of this idea was obtained using mutations at positions 45 and 67. Introduction of a negative charge at these positions dramatically increases the rate of autooxidation while introduction of a positive charge inhibits autooxidation.

Scheme I1 points out the close mechanistic relationship between autooxidation and oxygen affinity and the difficulty of constructing stable hemeproteins with low oxygen affinities. The hydrogen bond in native myoglobin decreases the rate of autooxidation while raising oxygen affinity. In order to raise the P5,, of myoglobin, either the hydrogen bonding interaction should be weakened or steric hindrance of the bound ligand should be enhanced. In single mutants, both these effects cause substantial increases in k,, (i.e. H64Q and V68I myoglobins in Tables I1 and 111, respectively). Thus, current attempts to engineer hemoglobin-based blood substi- tutes with lower affinities for oxygen must take into account the resulting detrimental effects on stability to autooxidation.

APPENDIX A

Time Dependence of [OJ at Low Levels Regardless of the exact mechanism, the overall stoichiom-

etry for the autooxidation of myoglobin is a net consumption of 0.25 mol of O2 per mol of myoglobin oxidized (Brown and Mebine, 1969):

[MbOz Mb + 0 2 1 + H+ 2 Mb+ + - Hz0 + - 0 2 (Eq. 1A) 1 3 2 4

As autooxidation proceeds in a stoppered cuvette or a closed tonometer, the concentration of free oxygen and the fractional

- 2

[Oz,] = [Oztotl - Y.[Mbtotl (Eq. 8A)

where [OPtot] is the total dioxygen concentration; [MbtOt], the total reduced heme concentration; [OZf,,], the free dissolved dioxygen concentration; and Y, the fractional saturation of reduced myoglobin with oxygen.

The magnitude and direction of the change in free [O,] depends on the particular protein and the experimental conditions. In principle, the small amount of dissolved oxygen in the autooxidation solution is buffered by the much larger concentration of oxygen in the gas phase above the solution, even in small stoppered 3-ml cuvettes. However, the solutions were not stirred because of the long incubation times. Calcu- lations of the time dependence of free and total oxygen concentration in samples were made assuming no buffering by the gas phase to estimate the upper limit of these changes. In general, the consumption of oxygen is insignificant for autooxidation measurements at concentrations of dissolved oxygen around 50 p~ or higher when the protein concentration was kept a t 520 p~ heme. Even at lower levels, the changes in free oxygen concentration are small. For example, in the case of the H64V mutant, the Kd for O2 binding is very large and the protein is primarily deoxymyoglobin in 1% 02- equilibrated buffer. Under these conditions, the oxidation of 20 p~ H64V protein results in a decrease in free [O,] from -12 to -9 p~ after 3 half-lives, due to the consumption of oxygen by the oxidation of deoxymyoglobin. In contrast, when 20 p~ native sperm whale myoglobin is oxidized under the same conditions, the protein starts out as oxymyoglobin and the free [02] increases from -12 to -20 pM after 3 half-lives, due to the release of oxygen originally bound to the reduced protein.

APPENDIX B

Derivation of Autooxidation Rate Equations Macroscopic Rate Equations for the General Scheme-When

both the uni- and bimolecular mechanisms for autooxidation apply, the following chemical reaction scheme may be written:

+ N 2 MbN + 0 2 - Mb+ + 0, b

k-I

where Mb02, Mb, and Mb' represent oxy (Fe(II)), deoxy (FeII)), and met (Fe(II1)) myoglobin and N is the facilitating nucleophile for the bimolecular mechanism (see Equations 1


and 3-5 and Scheme I). The rate of formation of ferric myoglobin is given by:

” ‘IMbf1 - k2[O2][MbN] + kdj.s[h4bOzl (Eq. 10A) d t

The concentrations of MbN and MbO, can be derived in terms of the total amount of ferrous myoglobin remaining, Mbbt, by invoking a steady-state assumption for MbN, assuming MbOz and Mb are in rapid equilibrium, and considering mass balance:

” d[MbN1-O=k,[N][Mb]-k-l[MbNJ-kz[Oz][MbN] (Eq. 11A) dt

(Eq. 12A)

[Mb,] = (Mb] + [MbOz] + [MbN] (Eq. 13A)

In their original analysis, Wallace et al. (1982) argued that the [MbN] term in the mass balance equation could be ne- glected since no spectral intermediates indicative of a 6- coordinate complex were observable. Under these conditions,

(Ea. 14A)

Substituting these expressions into Equation 10A and noting that (d[Mb&dt) = (-d[Mb+]/dt) gives:

and the observed pseudo-first order autooxidation rate constant, k,,, is given by Equation 11 under “Results.”

If the [MbN] term in the mass balance equation is retained, the expressions for [MbN] and [MbOz] become significantly more complex:

Substitution into Equation 1OA gives the following rate equation:

(Eq. 18A)

This expression only applies when [MbN] is greater than 10% of the total ferrous forms present ( i e . only a t low [OZ] when

Equation 18A predicts that k, should exhibit a hyperbolic dependence on [N] if the nucleophile concentration can be made extremely high. As [N] + m, the observed rate constant will approach kz [02] and the process should be completely bimolecular. Both Wallace et al. (1982) using hemoglobin and Satoh and Shikama (1981) using myoglobin reported a linear dependence on the concentration of azide and other facilitating nucleophiles. In our experiments, deviations from linearity were observed, particularly at low O2 concentrations (Fig. 6A).

Interpretations in Terms of Microscopic Constants-In Scheme I1 of the main text, Mb02 is partitioned into three

kl [N] 2 0.1k-1).

microscopic species B, C, and A representing Fe(I1). 0, with a hydrogen bond to HisM, Fe(I1) .02 without a stabilizing hydrogen bond, and the protonated form Fe(I1) -02H+, respectively. This mechanism provides expressions for the macroscopic equilibrium and rate constants, Kd and kdiss, in terms of: 1) the microscopic hydrogen bonding parameter Kg; 2) the acid dissociation equilibrium constant for Fe(I1) .OzH+, KA; 3) the intrinsic equilibrium constant for 0, dissociation from species C, K’d; and 4) the intrinsic rate constant for HOz dissociation from species A, k’diss .

For example, the fractional saturation of ferrous myoglobin with O2 is defined as:

and the relationship between the intermediates in Scheme I1 are:

Defining the relative concentration of C as I and solving for the relative concentrations of the remaining species, Y becomes:

and the observed Kd is given by:

(Eq. 21A)

(Eq. 22A)

where fc is the fraction of oxymyoglobin which is present as intermediate C in Scheme 11. Similar arguments can be used to show that:

(Eq. 23A)

Substitution of these expressions for Kd and kdiss into either Equation 11 of the main text (assuming [MbN] z= 0) or the more general Equation 18A gives the dependence of kox on the strength of the His64 hydrogen bond and the acid dissociation constant for Fe(11). 02H+.

Structural Interpretations of the MbN Intermediate-In the absence of added nucleophiles such as azide, we and Wallace et al. (1982) have assumed that the MbN intermediate represents a coordinated complex between water and Fe(I1). In all the known crystal structures of deoxymyoglobins containing His64 (native sperm whale myoglobin, Phillips, 1981; recombinant V68T pig myoglobin4; recombinant wild-type, V68A, and V68F sperm whale myoglobins5) there is a water molecule hydrogen-bonded to the-N, of the distal histidine. This water molecule is roughly 3.0 A away from the center of the porphy- rin ring and too far away to be considered 5oordinated to the reduced iron atom, which itself is 0.3-0.5 A below the plane of the pyrrole nitrogens. The occupancy of this noncoordi- nated water molecule is roughly one, suggesting strongly that water can readily enter the distal pocket of deoxymyoglobin. Thus, if HzO is the nucleophile that facilitates the bimolecular

~~ ~

A. D. Cameron and A. J. Wilkinson, unpublished data. M. L. Quillin and C. N. Phillips, Jr., unpublished data.


reaction pathway, k1 is given by the product of an equilibrium constant for partitioning from the solvent into the distal pocket (and attachment to His64, if present) times the rate constant for binding directly to the iron atom. The latter process must be slow, and the nature of the required structural changes, if any, are not clear.

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The Mechanism of Autooxidation of Myoglobin* · 2001-06-21 · toward the CD corner. iments for determining the mechanism of autooxidation are presented under "Results." MATERIALS