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THE JOURNAL 1992 by The American Society for Biochemistry OF BIOLOGICAL CHEMISTRY and Molecular Biology Inc Vol. 267, No. 32, Issue of November 15, PP. 22843-2285’2,1992 Printed in U.S.A. Coordination Structure of the Ferric Heme Iron in Engineered Distal Histidine Myoglobin Mutants* (Received for publication, June 11, 1992) Masao Ikeda-Saito$$, Hiroshi Horill, Laura A. AnderssonII , Roger C. Prince**, Ingrid J. Pickering**, Graham N. George**, Charles R. Sanders II$, Rodney S. Lutz$, Erin J. McKelveyS, and Rafael Mattera$ From the $Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4970, the 7Department of Biophysical Engineering, Faculty of Engineering Science, Osaka University, Toyonaka, Osaka 560. JaDan. the ItDeDartment of Biochemistrv. Kansas State University, Manhattan, Kansas 66505, and **EmonResearch and I . , Engineering Company, Anna&ale, New Jeriey 08801 Recombinanthuman myoglobin mutantswiththe distal His residue (E7, Hiss4) replaced by Leu, Val, or Gln residues were prepared by site-directed mutagen- esis and expression in Escherichia coli. Electronic and coordination structures of the ferric heme iron in the recombinant myoglobin proteinswereexamined by optical absorption, EPR, ‘H NMR, magnetic circular dichroism, and x-ray spectroscopy. Mutations, His + Val and His + Leu, remove the heme-bound water molecule resulting in a five-coordinate heme iron at neutral pH, while the heme-bound water molecule ap- pears tobe retained in the engineered myoglobin with His + Gln substitution as in the wild-type protein. The distal Val and distal Leu ferric myoglobin mutants at neutral pH exhibited EPR spectra with g, values smaller than 6, which could be interpreted as an ad- mixture of intermediate (S = 3/2) and high (S = 5/2) spin states. At alkaline pH, the distalGln mutant is in the same so-called “hydroxy low spin” form as the wild- type protein, while the distal Leu and distal Val mu- tants are in high spin states. The ligand binding prop- erties of these recombinant myoglobin proteins were studied by measurements of azide equilibrium and cy- anide binding. The distal Leu and distal Val mutants exhibited diminished azide affinity and extremely slow cyanide binding, while the distal Gln mutant showed azide affinity and cyanide association rate constants similar to those of the wild-type protein. * This work was supported by National Institutes of Health (NIH) Grants GM39492 (to M. 1.-S.), GM39359 (to M. 1.-S.), GM47485 (to C. R. S.), Biomedical Research Support Grant RR05410-28 (to R. M.), grant-in-aids from the Northeast Ohio Affiliate of the American Heart Association (to M. I.-%) andtheKansas American Heart Association KS-92-GB-1 (to L.A. A.), Kansas Agricultural Experi- mental Station (contribution no 92-597-5) (to L. A. A.), atravel grant from the Yamada Foundation (to H. H.), and a Grant-in-Aid for Scientific Research in Priority Area 04225221 and a Research Grant 04680270 from the Japanese Ministry of Education, Science and Culture (to H. H.). The purchases of the stopped-flow spectro- photometer and the Bruker EPR instrument were in part supported by an NIH Small Instrumentation Grant (to M. I.-S.) and by NIH Grant RR05659 (to M. 1.-S.), respectively. A portion of the work was done at Stanford Synchrotron Radiation Laboratory (SSRL), which is operated by theDepartment of Energy, Division of Chemical Science. TheSSRL Biotechnology Program is supported by the Biomedical Resource Technology Program, National Center of Re- search Resources, NIH. 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. I To whom correspondence should be addressed. The coordination and electronic structures of heme iron in ferric hemoproteins andheme model systems have been the subjects of various spectroscopic studies. Most ferric Mbs’ and Hbs possess a heme iron in the six-coordinate high spin state with a water molecule as a dissociable axial ligand (Peisach et al., 1984). The bound water molecule forms a hydrogen bond with the invariant distal His resi- due (His64, E7), which is located in close proximity to the ligand binding site (Antonini and Brunori, 1971). Globins lacking the distal His show spectroscopic and ligand bind- ing propertiesdifferent from those of typical Mb. For example, the light absorption spectra of ferric Aplysia Mb and Glycera Hb (Giacometti et al., 1981; Mintorovitch and Satterlee, 1988) are significantly different, both in extinc- tion and maxima, from those of typical ferric Mbs and Hbs. Ferric Aplysia Mb (distal His + Val) binds azide with much lower affinity than sperm whale or horse Mb (Giacometti et al., 1975). The cyanide binding properties of ferric Glycera Hb (distal His + Leu) have been reported to be anomalously slow when compared to ferric Mb with distalHis(MintorovitchandSatterlee, 1988). In ferric Aplysia Mb the sixth coordination position of the heme iron is vacant (Bolognesi et al., 1989), and the optical spectrum suggests that this is also the case in Glycera Hb (Giacometti et al., 1981; Mintorovitch and Satterlee, 1988; Arents and Love, 1989). On the other hand,ferric elephant Mb (distal His + Gln) showed optical spectral properties similar to those of typical ferric Mb (Bartnicki et al., 1983), and the sixth coordination position of the heme iron in ferric elephant Mb was considered to be occupied by a water ligand (Bartnicki et al., 1983; Krishnamoorthi et al., 1984). These results suggest that the nature of the E7 residue could dictate the coordination and reactivity of the ferric heme iron Mb. Recent development in proteinengineering technologies have made it possible to obtain mutant Mbs and Hbs in the large quantities necessary for structural and functional studies (Nagai and [email protected], 1984; Varadarajan et al., 1985; Springer and Sligar, 1987). Considerable advances have been attained in understanding how amino acid res- idues in the distal heme pocket control ligand binding to the ferrous Mb through the extensive kinetic measure- ments on Mb mutants (Rohlfs et al., 1990; Carver et d., 1990). The physiological role of the invariant distal His in ligand binding to the ferrous sperm whale Mb is now The abbreviations used are: Mb(s), myoglobin(s); Hb, hemoglo- bin; MES, 2(N-morpholino)ethanesulfonic acid; MCD, magnetic cir- cular dichroism. 22843

Coordination Structure the Ferric Heme Iron in Engineered ... · The heme iron coordination structures at cryogenic temperatures were examined by iron x-ray absorption edge spectroscopy,

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  • THE JOURNAL 1992 by The American Society for Biochemistry

    OF BIOLOGICAL CHEMISTRY and Molecular Biology Inc

    Vol. 267, No. 32, Issue of November 15, PP. 22843-2285’2,1992 Printed in U.S.A.

    Coordination Structure of the Ferric Heme Iron in Engineered Distal Histidine Myoglobin Mutants*

    (Received for publication, June 11, 1992)

    Masao Ikeda-Saito$$, Hiroshi Horill, Laura A. AnderssonII , Roger C. Prince**, Ingrid J. Pickering**, Graham N. George**, Charles R. Sanders II$, Rodney S . Lutz$, Erin J. McKelveyS, and Rafael Mattera$ From the $Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4970, the 7Department of Biophysical Engineering, Faculty of Engineering Science, Osaka University, Toyonaka, Osaka 560. JaDan. the ItDeDartment of Biochemistrv. Kansas State University, Manhattan, Kansas 66505, and **Emon Research and

    I . ,

    Engineering Company, Anna&ale, New Jeriey 08801

    Recombinant human myoglobin mutants with the distal His residue (E7, Hiss4) replaced by Leu, Val, or Gln residues were prepared by site-directed mutagen- esis and expression in Escherichia coli. Electronic and coordination structures of the ferric heme iron in the recombinant myoglobin proteins were examined by optical absorption, EPR, ‘H NMR, magnetic circular dichroism, and x-ray spectroscopy. Mutations, His + Val and His + Leu, remove the heme-bound water molecule resulting in a five-coordinate heme iron at neutral pH, while the heme-bound water molecule ap- pears to be retained in the engineered myoglobin with His + Gln substitution as in the wild-type protein. The distal Val and distal Leu ferric myoglobin mutants at neutral pH exhibited EPR spectra with g, values smaller than 6, which could be interpreted as an ad- mixture of intermediate ( S = 3/2) and high ( S = 5/2) spin states. At alkaline pH, the distal Gln mutant is in the same so-called “hydroxy low spin” form as the wild- type protein, while the distal Leu and distal Val mu- tants are in high spin states. The ligand binding prop- erties of these recombinant myoglobin proteins were studied by measurements of azide equilibrium and cy- anide binding. The distal Leu and distal Val mutants exhibited diminished azide affinity and extremely slow cyanide binding, while the distal Gln mutant showed azide affinity and cyanide association rate constants similar to those of the wild-type protein.

    * This work was supported by National Institutes of Health (NIH) Grants GM39492 (to M. 1.-S.), GM39359 (to M. 1.-S.), GM47485 (to C. R. S.), Biomedical Research Support Grant RR05410-28 (to R. M.), grant-in-aids from the Northeast Ohio Affiliate of the American Heart Association (to M. I.-%) and the Kansas American Heart Association KS-92-GB-1 (to L. A. A.), Kansas Agricultural Experi- mental Station (contribution no 92-597-5) (to L. A. A.), a travel grant from the Yamada Foundation (to H. H.), and a Grant-in-Aid for Scientific Research in Priority Area 04225221 and a Research Grant 04680270 from the Japanese Ministry of Education, Science and Culture (to H. H.). The purchases of the stopped-flow spectro- photometer and the Bruker EPR instrument were in part supported by an NIH Small Instrumentation Grant (to M. I.-S.) and by NIH Grant RR05659 (to M. 1.-S.), respectively. A portion of the work was done a t Stanford Synchrotron Radiation Laboratory (SSRL), which is operated by the Department of Energy, Division of Chemical Science. The SSRL Biotechnology Program is supported by the Biomedical Resource Technology Program, National Center of Re- search Resources, NIH. 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.

    I To whom correspondence should be addressed.

    The coordination and electronic structures of heme iron in ferric hemoproteins and heme model systems have been the subjects of various spectroscopic studies. Most ferric Mbs’ and Hbs possess a heme iron in the six-coordinate high spin state with a water molecule as a dissociable axial ligand (Peisach et al., 1984). The bound water molecule forms a hydrogen bond with the invariant distal His resi- due (His64, E7), which is located in close proximity to the ligand binding site (Antonini and Brunori, 1971). Globins lacking the distal His show spectroscopic and ligand bind- ing properties different from those of typical Mb. For example, the light absorption spectra of ferric Aplysia Mb and Glycera Hb (Giacometti et al., 1981; Mintorovitch and Satterlee, 1988) are significantly different, both in extinc- tion and maxima, from those of typical ferric Mbs and Hbs. Ferric Aplysia Mb (distal His + Val) binds azide with much lower affinity than sperm whale or horse Mb (Giacometti et al., 1975). The cyanide binding properties of ferric Glycera Hb (distal His + Leu) have been reported to be anomalously slow when compared to ferric Mb with distal His (Mintorovitch and Satterlee, 1988). In ferric Aplysia Mb the sixth coordination position of the heme iron is vacant (Bolognesi et al., 1989), and the optical spectrum suggests that this is also the case in Glycera Hb (Giacometti et al., 1981; Mintorovitch and Satterlee, 1988; Arents and Love, 1989). On the other hand, ferric elephant Mb (distal His + Gln) showed optical spectral properties similar to those of typical ferric Mb (Bartnicki et al., 1983), and the sixth coordination position of the heme iron in ferric elephant Mb was considered to be occupied by a water ligand (Bartnicki et al., 1983; Krishnamoorthi et al., 1984). These results suggest that the nature of the E7 residue could dictate the coordination and reactivity of the ferric heme iron Mb.

    Recent development in protein engineering technologies have made it possible to obtain mutant Mbs and Hbs in the large quantities necessary for structural and functional studies (Nagai and [email protected], 1984; Varadarajan et al., 1985; Springer and Sligar, 1987). Considerable advances have been attained in understanding how amino acid res- idues in the distal heme pocket control ligand binding to the ferrous Mb through the extensive kinetic measure- ments on Mb mutants (Rohlfs et al., 1990; Carver et d., 1990). The physiological role of the invariant distal His in ligand binding to the ferrous sperm whale Mb is now

    The abbreviations used are: Mb(s), myoglobin(s); Hb, hemoglo- bin; MES, 2(N-morpholino)ethanesulfonic acid; MCD, magnetic cir- cular dichroism.

    22843

  • 22844 Ferric Mb Mutants

    established (Carver et al., 1990): the polarity of the imid- azole side chain is required to stabilize bound 0, by hydro- gen bonding, although it inhibits the rate of the entry into the distal pocket of apolar ligand. From the overall ligand binding properties of the ferrous sperm whale Mb with distal His mutations, Rohlfs et al. (1990) suggested that the aliphatic nature of the side chain of the E7 Leu in Glycera Hb was responsible for the lowered 0, affinity and the large CO association rate. The functional properties of ferrous Aplysia Mb could not be explained solely by the presence of Val at the E7 site (Rohlfs et al., 1990; Cutruz- zola et al., 1991). The Fez+. 0, complex in sperm whale Mb containing Gln'j4 is stabilized, but the magnitude of the stabilization is much smaller than that in elephant Mb (Rohlfs et al., 1990). The different functional properties of these naturally occurring proteins with Val, Leu, and Gln at the E7 site thus cannot be due solely to the nature of the distal E7 residue. For the ferric Mb derivatives, the effect of mutation at the distal His on the spin and coordination states has been studied by optical absorption, resonance Raman, NMR, and azide binding (Morikis et al., 1990; Rajarathnam et al., 1991; Cutruzzola et al., 1991). On the basis of Soret absorption band and Raman porphy- rin core-size marker bands, Morikis et al. (1990) reported that the replacement of the distal His by Val, Met, or Phe removes the ligand water molecule from the sixth coordi- nation position of the heme iron in ferric sperm whale Mb resulting in the five-coordinate heme species, while the ligand water molecule is retained in the ferric Mb mutants with Gly or Ala at the E7 site. Though to a lesser extent, single substitution of Val for His at the E7 site destabilizes the Fe3+. N3 complex in ferric sperm whale Mb as seen in ferric Aplysia Mb (Cutruzzola et al., 1991), indicating that the ligand binding properties of the Aplysia Mb could be somewhat qualitatively mimicked by the substitution of the distal His by Val in sperm whale Mb. Hyperfine-shifted proton magnetic resonance studies have been performed on ferric sperm whale Mb mutants with the His "-* Gly, His -+ Val, His + Phe, and His + Gln mutations at the E7 position and other E l l mutants (Rajarathnam et al., 1991). On the basis of the hyperfine shift pattern, Rajar- athnam et al. (1991) suggested that substitution of the distal His by Val or Phe abolishes the water coordination and that the replacement by Gln or Gly leads to a fractional water coordination. They concluded that the heme iron is predominantly five coordinate in ferric Mb, with Gln or Gly at the E7 site, and in ferric elephant Mb (E7 Gln). The proton magnetic resonance conclusions on the Gly and Gln mutants conflict not only with the previous NMR and Raman results (Krishnamoorthi et al., 1984; Morikis et al., 1990) but also with those of the x-ray crystallo- graphic structural determination of those Mb mutants, which indicated water coordination in ferric Mb mutants with Gly and Gln at the E7 site (Quillin et al., 1992).

    Our objectives of this paper are %fold. One is to precisely assess the possible relationship between the iron coordi- nation structure, reactivity of the ferric heme iron, and the nature of the distal residue at the E7 site, and another is to resolve the current conflict on the coordination state of the ferric iron in the distal His -+ Gln mutant. In order to achieve these goals, we have expressed (in Escherichia coli) three mutant Mb fusion proteins in which Val, Leu, or Gln residues replace the E7 distal His of the wild-type protein, using the human Mb expression system reported by Varadarajan et al. (1985). We chose these substitutions, because Leu, Val, and Gln residues are found at the distal

    His site of Glycera Hb, Aplysia Mb, and elephant Mb, respectively (Dickerson and Geis, 1983), so that the pres- ent mutant ferric Mb results could be compared with those of the naturally occurring proteins. The water coordination to the heme iron in these recombinant Mb at room tem- perature was inferred from the light absorption and MCD spectra on the basis of the data reported for hemoproteins and model systems with known coordination structure: five-coordinate high spin hemoproteins exhibit broad So- ret absorption bands around 400 nm with an extinction coefficient of around 100 mM" cm-l, while six-coordinate high spin species have sharp Soret bands near 409 nm (Giacometti et al., 1981). The five-coordinate hemopro- teins exhibit relatively weak and asymmetric signals in the Soret region of the MCD spectra, while strong and sym- metric derivative-shaped Soret MCD signals are seen in six-coordinate ferric high spin species (Bracete et al., 1991). The heme iron coordination structures at cryogenic temperatures were examined by iron x-ray absorption edge spectroscopy, which discerns five- and six-coordinate ferric high spin heme iron derivatives (Shiro et al., 1990). The electronic structure of the heme iron in these recombinant human Mb was studied by EPR spectroscopy. Hyperfine- shifted proton magnetic resonance of ferric Mb recombi- nants were also recorded so as to assess the relationship between the hyperfine shift pattern and the iron coordi- nation structure of the ferric heme iron. In addition the effects of distal His mutation on the cyanide and azide binding properties were also determined.

    EXPERIMENTAL PROCEDURES

    The human Mb expression system was a kind gift of Dr. S. G. Boxer (Department of Chemistry, Stanford University). Induction of fusion proteins in E. coli, AR68, and purification of Mb were carried out as reported by Varadarajan et al. (1989). The standard reference protein, H64H, in which C ~ S " ~ has been replaced by Ala for ease in purification was originally designated as CllOA' (Varadarajan et al., 1989). The spectroscopic and functional properties of this mutant were indistinguishable from those of the wild-type Mb (Varadarajan et al., 1989), and we therefore designate CllOA as the wild type for purposes of discussion. All DNA manipulations were as described in Sambrook et al. (1989). Oligonucleotides necessary for mutagenesis and DNA sequencing were provided by Dr. S. Kimura (National Cancer Institute). Site-directed mutagenesis was carried out using an Amersham oligonucleotide-directed in vitro mutagenesis kit following the manufacturer's instructions. Mutations were confirmed by DNA sequencing by the use of a United States Biochemical Sequenase kit.

    Optical spectra were recorded with a Hitachi U-3210 instrument at 20 "C. Azide equilibrium measurements were performed by the addition of a known amount of sodium azide solution to the Mb solution followed by optical measurements in the Soret region as described previously (Ikeda-Saito, 1985). Fractional saturation was calculated from the absorbance difference induced by ligand binding. All ligand equilibrium data in the present study followed n = 1 titration curves. Three to four measurements were performed under each set of experimental conditions. Cyanide binding was studied by the rapid-mixing method using a Hi-Tech SF-51MX stopped-flow spectrophotometer under pseudo-first order conditions. Mb (3 pM) was mixed with cyanide (between 5 and 250 mM), and the time course of the formation of the cyanide complex was followed by the absorb- ance change a t 409 nm (for H64H and H64Q) or at 395 nm (for H64V and H64L). All kinetics were of single exponential character, and the reaction rates were estimated by iterative least squares fitting over 400 data points. Under these condition, the observed rate was linearly proportional to the cyanide concentrations, and rate saturation was not observed. Three to four measurements were performed under each set of experimental conditions. Measurements of x-ray absorp-

    ' The naming convention for myoglobin mutants followed Vara- darajan et al. (1989). H64H, H64L, H64V, and H64Q correspond to the human Mb proteins with His, Leu, Val, and Gln at the 64th amino acid position (the E7 distal histidine site).

  • Ferric Mb Mutants 22845

    tion spectra were carried out at the Stanford Synchrotron Radiation Laboratory on bean line 7-2 using a Si (220) double crystal mono- chromator. Four to six 30-min scans were averaged, and samples were maintained at a temperature close to 5 K in an Oxford Instruments CD204 flowing liquid He cryostat during measurements. X-ray ab- sorption spectra were measured as the fluorescence excitation spectra using a Canberra 13-element detector (Cramer et al., 1988). The extended x-ray absorption-fine structure oscillations were quantita- tively analyzed as previously described (George et al., 1989) using theoretical curve-wave phase and amplitude functions calculated with the program feff of Rehr and co-workers (Rehr et al., 1991; Mustre de Leon et al., 1991). The data were smoothed by convolution with a Gaussian function of half-width 0.1 A" prior to the fit, and only the first shell of scatterers was fitted. The x-ray energy was calibrated with respect to the first inflection energy of an iron metal foil, which was defined as 7111.3 eV. EPR spectra were recorded on a Bruker ESP-300 instrument operating at 9.45 GHz. Experiments were carried out at an incident microwave power of 1 milliwatt with a field modulation of 1 mT at 100 kHz. An Oxford flow cryostat (ESR-900) was used for liquid helium temperature measurements. Microwave frequency was monitored by a Hewlett-Packard 5350B frequency counter, and a Bruker ER-035M NMR Gauss meter was used to determine the magnetic flux density. MCD spectra were obtained on a Jasco 5-720 spectropolarimeter with a JASCO electromagnet. Meas- urements were carried out at room temperature under a magnetic flux density of 1.39 tesla with the magnetic field direction parallel to the direction of light propagation. Light aborption spectra of the Mb samples were measured before and after MCD/CD measurements, and in no case was a spectral change greater than 2% observed following MCD/CD examination. The spectropolarimeter was cali- brated for intensity with ammonium d-10-camphor sulfonate and for magnetic flux density with a freshly prepared ferricyanide solution. 'H NMR spectra were obtained using a Bruker AC270 spectrometer at 25 "C. Spectra were produced from 16 K points obtained over 9000- Hz windows following a 6-ps 45" pulse. Samples were in deuterated 0.1 M phosphate buffer, pH* 6 (uncorrected pH meter reading). The residual water peak was suppressed by a 0.2-s presaturation pulse, and the signal-to-noise ratios were improved by 10-Hz line broaden- ing. The chemical shifts were referred to HDO in ppm.

    Based on the spectroscopic properties of recombinant human Mb, Varadarajan et al. (1989) concluded that the coordination structure of the heme group in ferric human recombinant Mb is essentially the same as that of sperm whale Mb. As shown below, our present MCD, NMR, and EPR data further support their conclusions: ferric human Mb is a high spin complex with a water molecule as its sixth ligand at neutral pH, and its alkaline form is a low spin complex with an - OH ligand, as established in the better characterized authentic and recombinant sperm whale Mb proteins (Antonini and Brunori, 1971; Blumberg and Peisach, 1971; Peisach et al., 1984; Morikis et al., 1990; Phillips et al., 1990).

    RESULTS

    Fig. 1 illustrates the light absorption spectra of ferric recom- binant Mb in 0.1 M phosphate buffer, pH 6 (A , left panel) and in 0.1 M Glycine-NaOH buffer, pH 10 (B, right panel) at 20 "C. The spectra of H64L and H64V are very similar, and Fig. 1 reports only that of H64L for clarity. The wild-type protein (H64H) exhibits a spectrum similar to sperm whale met Mb, in agreement with the original report of Varadarajan et al. (1989). Ferric Mb H64V and H64L exhibited optical absorption spectra quite different from that of the wild-type: the Soret peaks are broad and shifted toward higher energy, and the Soret extinction coefficients are smaller. Indeed, the spectral features of H64V and H64L at pH 6 are similar to those of Glycera Hb component 11, Aplysia Mb, and horse- radish peroxidase (Giacometti et d . , 1981; Mintorovitch and Satterlee, 1988) which are known to have a vacant sixth coordination position (Vuk-Pavlovic and Siderer, 1977): the light absorption spectra are indicative of five-coordinate high spin heme iron in H64L and H64V in agreement with the earlier conclusions on sperm whale H64V (Morikis et al., 1990). The optical spectral properties of ferric Mb H64Q are similar to those of H64H, but differences are noticed between these two spectra; there is a shoulder on the blue side of the Soret band (around 380 nm), and a 4-nm red shift of the so- called charge transfer band to 637 nm upon His + Gln mutation. Absorption maxima and extinction coefficients of the recombinant Mb at pH 6 are listed in Table I.

    At pH 10, H64Q shows an optical spectrum of hydroxide- type low spin as does the wild-type protein (Varadarajan et al. 1989): the heme iron assumes a low spin state with an - OH group as its sixth ligand (Antonini and Brunori, 1971). The pH-dependent spectral changes were reversible between pH 6 and 11. The pKa values of this acid-base transition for the wild-type and H64Q were determined as 8.6 and 8.1, respectively. The optical spectra of H64V and H64L are essentially independent of pH between pH 6 and 9. At pH 10, the original broad Soret peak at 398 nm is replaced with two peaks at 375 and 404 nm with the spectral shape in the visible region indicative of ferric high spin state (Fig. lB) , quite different from the low spin spectra seen for the wild-type and H64Q. The pK, values of this transition to the alkaline high spin form were found to be about 10, but we were not able to precisely determine them because of the appearance of an- other species above pH 12. The optical spectrum of Glycera

    FIG. 1. Light absorption spectra of ferric recombinant Mb in 0.1 M phosphate buffer, pH 6 (left panel), and 0.1 M glycine-NaOH buffer, pH 10 (right panel).

    I 50

    IO0

    50

    0

    150 f L

    pH 10

    400 500 600 700 400 500 600 700

    w a v e l e n g t h ( n m )

  • 22846 Ferric Mb

    ferric Hb was reported to be independent of pH between pH 6 and 9 (Mintorovitch and Satterlee, 1988), which agrees with the results on H64L. Aplysia ferric Mb was reported to exhibit an acid-base transition with a pK, value of 7.6 (Antonini and Brunori, 1971). Thus, ferric H64V showed pH-dependent spectral changes different from Aplysia Mb.

    Fig. 2 shows the MCD spectra of H64H, H64Q, H64V, and H64L in 0.1 M phosphate buffer, pH 6. The MCD spectra of H64H and H64Q, with a sharp derivative-shape peak in the Soret region, are very similar to those of six-coordinate ferric high spin hemoproteins such as sperm whale Mb (Vickery et al., 1976; Nozawa et al., 1976). With diminished Soret features, the MCD spectra of H64L and H64V are distinctly different from those of the wild-type and H64Q, but the MCD spectral properties, such as intensity, position, and spectral shape, are very similar to those of horseradish peroxidase (Nozawa et al., 1976) and Aplysia Mb (Bracete et al., 1992) which are known to contain no water at the sixth coordination site (Vuk-Pavlovic and Siderer, 1977; Bolognesi et al., 1989).

    Ferric hemoproteins are known to bind small ionic ligands (Antonini and Brunori, 1971). Ferric Mb H64H and H64Q bind cyanide and imidazole to form low spin complexes, the optical spectra of which are very similar to those of sperm

    TABLE I Light absorption maxima (in nanometers) and extinction coefficients

    of the Soret band of ferric recombinant human Hb in 0.1 M phosphate buffer, pH 6.0, at 20 "C

    Protein Soret f a Visible mM' cm"

    H64H (wild type) 409.5 (153) 504 633 H64Q 409' (127) 506 637 H64V 395 (103) 507' 641 H64L 395 (103) 507d 641 Extinction coefficients were determined by the pyridine hemo-

    chromogen method (Antonini and Brunori, 1971). * Shoulder at -390 nm.

    Shoulder at -543 nm. Shoulder at -543 nm.

    H64H, H64Q, H64L, and H64V. FIG. 2. MCD spectra of ferric Mb

    Measurements were carried out in 0.1 M phosphate buffer, pH 5.6.

    3

    'F i E 0

    Mutants

    whale and horse Mb complexes (Antonini and Brunori, 1971). While H64H and H64Q form high spin fluoride complexes, addition of a saturating amount of fluoride to H64L and H64V in 0.1 M phosphate buffer, pH 7, at 20 "C did not cause spectral changes, indicating diminished fluoride affinity of H64V and H64L under these conditions. It should be pointed out that Aplysia Mb forms a fluoride complex (Bolognesi et al., 1990). Addition of azide yielded a predominantly low spin species for all four recombinant proteins similar to that seen for sperm whale Mb (Antonini and Brunori, 1971).

    The ligand binding properties of the human Mb recombi- nants were studied by measurements of cyanide and azide binding. Fig. 3A shows the sodium azide equilibrium curves measured at pH 7. Azide affinity decreases in the order H64Q (high affinity) > H64H >> H64V > H64L (low affinity) under these conditions. The dissociation constants of 25 p~ and 1.1 mM for the azide complexes of H64H and H64V of human Mb agree well with those of 27 p~ and 1.4 mM reported for the N3 complexes of H64H and H64V of sperm whale Mb (Cutruzzola et al., 1991).

    Since Glycera ferric Hb has been reported to exhibit anom- alously slow cyanide binding (ken = 5 X 10" M" s-') (Min- torovitch and Satterlee, 1988), we have conducted cyanide binding rate measurements for the four recombinant proteins. Fig. 3B illustrates the time course of the normalized absorp- tion change upon reaction of ferric Mb (3 C ~ M ) with potassium cyanide (25 mM) at pH 7. Similar experiments were carried out at seven different cyanide concentrations between 5 and 200 mM, and plots of the observed rate, kobs (s-'), against cyanide concentration were linear with linear correlation coef- ficients between 0.989 and 0.998 (data not shown). The cya- nide association rate constants, k,,, were estimated as 1.5 X IO2, 1.5 X lo2, 1.9 X 10, and 2.7 M" s-' for H64H, H64Q, H64V, and H64L, re~pectively.~

    Our analysis of the on constants followed the procedure of Min- torovitch and Satterlee (1988) in which "cyanide" represents the total amount of cyanide present (CN- and HCN), so that rate constants for Glycera Hb and human Mb mutants can be compared directly.

    30 - human metMb distal His mutant wild-type, HUH

    """ His -> Gln, H64Q

    . . . . . . . His -> Val, H64V ' \

    I \ 20 -

    v: ;

    -20 1 -30

    300 400 500 600 700 wavelength (nm)

  • Ferric Mb Mutants 22847

    A human M b (Fe ) + NaN,, pH7, 20 "C 3+

    0.0

    B h u m a n ferric Mb (3 pM) + KCN (25 mM) 1.0

    e,

    ? a c 0.8 e, 0 c a 0.6

    -El n v1 4 0.4 a e, N 3 .3

    z O 2 L 0

    0.0

    H 6 4 L

    H64V

    H 6 4 Q I . I , I , I ,

    0 1 2 3 4 5

    t ime ( s e c ) FIG. 3. A , azide equilibrium curves of ferric recombinant Mb pro-

    teins at pH 7 and 20 "C. Symbols are experimental data points, and the lines were drawn using the equation, y = [x]/(K., , + [ x ] ) , where Kapp, y,, and [ x ] are equilibrium dissociation constant, fractional saturatlon, and the ligand concentration, respectively, with the dis- sociation constants of 7, (H64Q), 25 (H64H), 1.1 X lo3 (H64L), and 1.6 X lo3 (H64V) p ~ , which were determined by iterative least squares fitting. B, the time course of the absorption change at the Soret peak associated with the formation of ferric Mb. CN complex. Symbols are experimental data points, and the least-squares fitted single-exponen- tial decay curve was drawn with the rates of 3.8, 3.8, 0.46, and 0.067 s-l for H64H, H64Q, H64V, and H64L, respectively.

    Fig. 4 shows the x-ray absorption spectra of the four Mbs under consideration here. I t is immediately apparent that they fall into two groups; H64H and H64Q look very similar, as do H64L and H64V. The spectrum of H64H is very similar to that of sperm whale Mb presented by Shiro et al. (1990). Shiro et al. (1990) have discussed the pre-edge feature at approximately 7112 eV as an indicator of the coordination state of the heme iron; an increase in its intensity indicates a

    decrease in the molecular symmetry around the iron atom. The relatively weak feature in H64H is consistent with the known presence of the water molecule at the six-coordination site. H64Q is clearly similar to H64H, thus it is likely to be six-coordinated, as indicated by the light absorption and MCD data. In contrast, the pronounced features in H64L and H64V indicate that the water molecule has been displaced and the iron is now five coordinate. The results of the extended x-ray absorption-fine structure analyses of the four proteins are shown in Table 11. As is typical of extended x-ray absorption- fine structure analyses, the estimates of the number of scat- terers, n, are subject to uncertainties of at least +30%, and so are not very informative in distinguishing five and six coor- dination of the iron atom. Nevertheless, the average bond length to scatterers, R, of similar atomic number can be determined with very high precision. The average Fe-N dis- tances are all in the range expected for high spin hemes.

    Fig. 5 shows the EPR spectra of the recombinant ferric Mb proteins in 0.1 M MES buffer, pH 6 (top panel), and in 0.1 M glycine-NaOH buffer, pH 10 (bottom panel). At pH 6, H64H shows an axial ferric high spin EPR spectrum which is essen- tially the same as that of acid met form of sperm whale Mb. Ferric H64Q exhibited a high spin EPR spectrum with a g 6 signal which seems to consist of two components: a sharp g = 5.95 axial high spin signal and a slightly rhombic high spin signal (marked by arrows in the figure) the g values of which could not be determined due to overlap with the large major axial high spin signal. The EPR spectra of H64L and H64V are quite different; they are rhombic, with gl = 6.13, g, = 5.62, g, = 2.0, and g, = 6.12, g, = 5.56, and g, = 2.0, respectively. The averaged g, values ((gl + g2)/2), 5.875 for H64V and 5.84 for H64L, are smaller than 6. One possible explanation for gL values less than 6 is the quantum mechanical admixture of S = 512 and 312 states proposed by Maltempo (1974). Relative contributions of high spin and intermediate spin states were estimated as described by Maltempo (1974): H64L is 92% S = 512 and 8% S = 312, and H64V is about 95% S = 512 and 5% S = 312 states. EPR spectra of ferric Glycera Hb were reported by Seamonds et al. (1974) and Hori et al. (1990). Although the spectrum at pH 7 reported by Seamonds et al. (1974) was a mixture of a nearly axial (gl = 6) and rhombic (g = 6.25,5.47, 2.0) species, Hori et al. (1990) showed that the spectrum consisted of two axial signals: one being a high spin species (S = 512, g, = 6) and the other an admixture of S = 512 and 312 (g = 5.62) species. Thus, the electronic structure of the heme iron in ferric H64L is different from that of Glycera Hb. The EPR spectrum of Aplysia Mb reported by Rotilio et al. (1971) looks like an axial symmetric ferric high spin spectrum at first sight, but was measured at 77 K where EPR spectra of rapidly relaxing species could become too broad to allow the recognition of rhombic high spin compo- nents. For example, under these conditions ferric H64V ex- hibited an axial high spin-type EPR spectrum with an ex- tremely broad g, signal with a peak-to-trough line width of 25 mT (data not shown) which could not be recognized as a rhombic high spin spectrum. The broad g, signal (g = 5.8) of the Aplysia Mb EPR spectrum reported by Rotilio et al. (1971) thus suggests a similar heme iron electronic structure in H64V and Aplysia Mb at neutral pH, but the lack of a 4.2 K EPR Aplysia Mb spectrum makes this only a tentative conclusion.

    In 0.1 M glycine-NaOH buffer, pH 10, H64H and H64Q exhibit low spin EPR signals typical of hydroxide-type he- moproteins (Blumberg and Peisach, 1971). In contrast, but as expected from the high spin-type light-absorption spectra (Fig. lB) , neither H64V and H64L exhibits an EPR signal with ferric low spin character. Instead, the spectra were

  • 22848

    FIG. 4. Fe K-edge x-ray absorp- tion spectra of ferric H64H, H64L, H64Q, and H64V recorded near 6 K.

    Ferric Mb Mutants

    i x 5 H64H

    H64L

    0

    H640

    D E

    H 6 4 V

    - I I I 1 I I I

    7 100 71 10 7120 7 130 7140 7 150 7160 7 170 Energy (eV1

    TABLE I1 Extended x-ray absorption fine structure curve-fitting results for human metmyoglobin distal His mutants

    The data were fitted over the range k = 1-16.2 A-', using theoretical curved-wave phase and amplitude functions calculated with the program feff. The values in parentheses are 95% confidence limits from the curve-fitting analysis and are given as an indication of the precision of the fit. The accuracy (as opposed to the precision) of the values determined in the fit are limited by the accuracy of the theory used to model the phase and amplitude functions. The confidence limits are thus a most optimistic estimate of the accuracies of the determined values. Typically, in extended x-ray absorption fine structure analyses, values for R (mean distance between the scatterers and the iron atom) are considered accurate within 0.02 A, and n (number of scatterers), uz (Debye-Waller factor), and A E O (change in the absorption edge energy dictated by the fitting) within 30 %.

    Protein n R 2 A A 2 eV

    H64H 4.79 (0.98) 2.0579 (0.0012) 0.00282 (0.00121) -9.22 (2.74) H64Q 3.96 (1.02) 2.0494 (0.0014) 0.00214 (0.00135) -9.01 (3.50) H64L 4.12 (0.95) 2.0390 (0.0012) 0.00207 (0.00120) H64V 4.50 (0.84) 2.0497 (0.0107) 0.00267 (0.00108)

    -8.81 (3.14) -8.05 (2.41)

    composites of at least two ferric high spin species; one essen- tially axial high spin (gl -6.05), the other rhombic high spin (g," = 6.67, g211 = 5.37, and g3I1 = 1.98). This is different from Glyceru Hb and Aplysiu Mb, where low spin EPR signals were dominant at alkaline pH (Seamonds et ul., 1972; Rotilio et ul., 1971).

    While the wild-type Mb exhibited the same EPR spectrum in 0.1 M HEPES, Tris, and phosphate buffers, pH 7, as in MES buffer, we found unexpected buffer-dependent spectral changes in H64V, H64L, and H64Q. Fig. 6, A-C, illustrates the EPR spectra of H64Q, H64V, and H64L recorded in MES, HEPES, Tris, and phosphate buffers, pH 7, respectively. In the case of H64L, the spectrum recorded in HEPES buffer (8, = 6.09, g, = 5.54, g3 = 2.0) is similar to that in MES buffer. The EPR spectra measured in phosphate and Tris buffers are almost superimposable, but they are quite different from those measured in MES and HEPES buffers. Although the H64L spectra in phosphate and Tris buffers seem to be almost axially symmetric at first glance, the line widths of the gl signals are wider than those of the typical axial signals seen in H64H and H64Q. Expanded recordings of the gL region

    indicated that these spectra are, in fact, best described as slightly rhombic with g, = 6.00 and g, -5.67. The gL value, as defined by (8, + g2)/2, is 5.85 both for the phosphate and Tris spectra. The measurements in phosphate or Tris showed smaller rhombicity without affecting the amount of interme- diate spin state mixed to the high spin state. The EPR spectra of H64V in Tris and phosphate buffers are very similar to each other, and they are slightly different from that in MES buffer: the spectra in MES and HEPES buffers are more anisotropic than those in Tris and phosphate buffers. In HEPES buffer, H64V exhibits two new EPR species, in addition to the rhombic signal seen in MES buffer (gl = 6.13, g, = 5.56, and g3 = 2.0). One is a rhombic high spin signal of which only the g, component is seen, at g = 6.35, since the g, component is overlapped with the g, signal of the major component (8, = 6.13, g, = 5.56, and g3 = 2.0). The other is a low spin signal with g, = 2.77, g2 = 2.29, g3 = 1.61. For H64Q, the spectrum in phosphate buffer is very similar to that in MES buffer, with an axial high spin signal (g, = 5.95 and gll = 2.0). The EPR spectrum in Tris buffer seems to be a rhombic high spin type with g, = 6.04, g2 = 5.94, and g3 = 2.0.

  • Ferric Mb Mutants 22849

    = p'" human Mb (Fe3+), 0.1 M MES, pH 6 9.46 GHz, 6 K

    g - 6.13

    9 = 5.97 9 = 2.00

    human Mb (Fe3+). 0.1 M Glycine-NaOH, pH 10

    9 = 6.05 9.46 GHz, 10 K

    A g = 1.96

    I g = 1.86 A. H64H

    kO.1 T+ 80" g +"r = 2.59 9 = 2.16 g = 1.84

    FIG. 5. EPR spectra of ferric H64H, H64Q, H64V, and H64L in 0.1 M MES buffer, pH 6 (top) and in 0.1 M glycine- NaOH buffer, pH 10 (bottom).

    H64Q in HEPES buffer exhibits both low spin and high spin species. There are a rhombic (gl = 6.29, g, = 5.53, with g3 not clearly defined) and an axial (gl = 5.95 and g,, = 2.0) high spin species, and two low spin species; one with g values of 2.77, 2.28 and 1.61, which appears to be the same low spin species seen for H64V in HEPES, pH 7, and the other with g values of 2.53,2.17, and 1,86, which is the same as the alkaline low spin of H64Q (spectrum B in Fig. 6B). Light absorption spectra were also recorded in these four buffers at 20 "C, and we found no buffer-dependent spectral change in the wild- type, H64Q, H64V, or H64L preparations.

    'H NMR spectra of ferric H64H (wild-type), H64Q, H64V, and H64L are shown in Fig. 7. Although the hyperfine-shifted patterns of the wild-type human Mb, H64Q, and H64V mu- tants are essentially the same as those of sperm whale Mb proteins reported by Rajarathnam et al. (1991), a difference is found in the spectrum of H64Q: the human Mb protein has an extra peak at 53 ppm which was missing in the sperm whale H64Q ~pect rum.~

    g = 6.06 human Mb (Fe3+1. H64V, pH 7

    L / 1 0 - 5.63 9.46 GHz, 6 K

    D. phosphate IY AI/ 1 1

    g - 5.95

    1 I

    human Mb LFe3+), H64a, pH 7

    9.46 GHz. 6 K

    g - 6.00 human Mb (Fe3+1, H64L pH 7 I

    9.46 GHz. 6 K

    D. phosphate jll I V

    v

    A. MES

    = I. t "o .1 T+ g - Loo 60 g - 6.54

    FIG. 6. EPR spectra of ferric Mb mutants in MES, HEPES, Tris, and phosphate buffers at pH 7.

    DISCUSSION

    Ferric Mb H64Q exhibits the sharp Soret MCD pattern typical of six coordinate ferric high spin heme comDounds (Nozawa et al., 1976; Vickery et a1.,-1976). Its light absorption

    The reason for this difference cannot be explained at this time. spectrum is slightly different from that of H64H in that the

  • 22850 Ferric Mb Mutants human m e w , distal His mutants, 0.1M phos., pH* 6, 25OC

    a a a n

    b Leu, H64L uii

    I UL

    b Val, H64V

    wild-type, H64H

    120 100 80 60 40 20 0 -20 -40 chemical &ifI from HDO (ppm)

    FIG. 7. 270 MHz 'H NMR spectra of ferric H64H, H64V, H64L, and H64Q in 0.1 M phosphate buffer, pH* 6 , recorded at 25 "C. Assignment of the peaks follows the established assignment on the sperm whale proteins (Rajarathnam et al., 1991): heme methyls (a) , propionate C,H ( b ) , F8 His CaH ( c ) , propionate CaH ( d ) , meso- H ( m ) , vinyl HPI ( q ) , and distal E7 methyl protons ( 2 ) .

    Soret extinction coefficient is smaller and there is a shoulder near 390 nm. This suggests that H64Q may be a mixture of five- and six-coordinate heme species with six-coordinate heme as the dominant species. The optical and EPR spectra of the alkaline form of H64Q are very similar to those of the alkaline hydroxyl form of ferric sperm whale Mb (Antonini and Brunori, 1971; Blumberg and Peisach, 1971), suggesting that an OH- is the sixth ligand at alkaline pH. The similar acid-base transition observed in H64H and H64Q argues for water coordination to the heme iron in H64Q, and the fact that the pK, value in H64Q is 0.5 pH unit lower than that of the wild-type (pK, 8.8) is in accord with the fact that the pK, value of ferric elephant Mb is about 0.5 pH unit less than those of most vertebrate Mb (Bartnicki et al., 1983). The His to Gln replacement at the E7 position thus stabilizes the coordinate hydroxyl ligand (Krishnamoorthi et al., 1984). We therefore conclude that the heme iron in ferric H64Q at neutral pH is likely to be a six-coordinate high spin species with a water molecule as the sixth ligand. The x-ray absorp- tion edge spectrum (Fig. 4) provides an elegant confirmation of this. Further support for this comes from the results of the x-ray structural determination of ferric sperm whale Mb H64Q which showed the presence of the ligand water molecule (Quillin et al., 1992).5 We note that this conclusion contradicts the very recent report by Rajarathnam et al. (1991) who inferred that H64Q and elephant Mb are predominantly five- coordinate species on the basis of the spectral patterns of hyperfine-shifted proton resonances of sperm whale Mb distal mutants. As mentioned above, our NMR spectra are essen- tially similar to those reported by Rajarathnam et al. (1991), with the exception of the additional peak at 53 ppm. Our current conclusion, in light of all the data presented here, is that the hyperfine shift pattern of 'H NMR spectra cannot be simply interpreted to provide a diagnostic of iron ligation in hemes.

    The light absorption spectra of ferric H64V and H64L in neutral pH are almost superimposable on those of ferric Aplysia Mb, cyanogen bromide (CNBr)-modified Mb, and horseradish peroxidase, which are known to contain no water

    G. N. Phillips, Jr., personal communication.

    at the sixth coordination site (Bolognesi et al., 1985; Bracete et al., 1992; Vuk-Pavlovic and Siderer, 1977); they are signif- icantly different, both in extinction and maxima, from those of hemoproteins that were shown to have a bound water as the sixth ligand (Giacometti et al., 1981). The MCD and x- ray absorption edge spectra of these two proteins are also consistent with five-coordinate heme iron in H64V and H64L (Nozawa et al., 1976; Shiro et al., 1990; Bracete et al., 1991). Upon the His + Leu and His + Val substitutions at the E7(64) distal site, the heme-bound water molecule is lost, resulting in the five-coordinate heme iron. This agrees with the Raman results of Morikis et al. (1990) on ferric sperm whale Mb H64V, and with those of x-ray crystallography on ferric sperm whale Mb H64V and H64L (Quillin et al., 1992).5 The loss of the bound water in ferric H64L and H64V may be attributable to inability to form a hydrogen bond to the distal base and/or hydrophobicity due to large nonpolar side chains in Leu and Val. At alkaline pH, ferric H64L and H64V are in the pure high spin state, in contrast to Glycera Hb and Aplysia Mb which were low spin compounds with an OH- group as the sixth ligand of the heme iron (Seamonds et al., 1972; Rotolio et al., 1970; Bolognesi et al., 1989). The absence of low spin hydroxy ligands in H64L and H64V cannot be simply due to the replacement of the distal His by Leu and Val, because these amino acids are the distal ligands in Aplysia and Glycera proteins. It therefore appears that amino acids other than the E7 residue are responsible for the binding of low spin hydroxy ligands to the sixth coordination positions of the ferric heme iron in the Glycera and Aplysia proteins.

    Both ferric H64V and H64L exhibited EPR spectra with slightly rhombic symmetry at neutral pH, while the EPR spectrum of H64H is axially symmetric. EPR spectra of ferric high spin hemoproteins with anionic axial ligands, such as cytochrome P-450, catalase, and peroxidases (Poulos, 1988), generally show rhombic symmetry (Peisach et aZ., 1971; Pal- mer, 1979), while the axial proximal His in ferric sperm whale Mb, which shows an axial high spin EPR spectrum, is less anionic (Palmer, 1979). In addition, the EPR spectra of five- coordinate Fe-imidazole (not imidazolate) tetraphenylpor- phyrin complexes show axial symmetry.'j In ferric H64L and H64V, the loss of the water ligand would disrupt the charge balance maintained at the ferric heme group in H64H, and the anionic character of the Fe-proximal His system could increase. This, in turn, could result in more anionic character of the proximal His in H64V and H64L, which would make their EPR spectra rhombic. The smaller degree of rhombicity seen in H64L and H64V than in the heme enzymes listed above could be due to the relatively weak imidazolate char- acter of the proximal ligand in these Mb mutants.

    The apparent g, values for H64L and H64V are smaller than 6 at neutral pH; the electronic structure of the heme iron in ferric H64L and H64V could not be described solely by the pure ferric high spin S = 5/2 state (Kotani, 1968). In this paper, we adopted the notion of admixture of S = 312 and S = 5/2 spin states developed by Maltempo to explain EPR g, values smaller than 6 observed in Chromatium cyto- chrome c' and horseradish peroxidase (Maltempo, 1974; Mal- tempo et al., 1975, 1979). Such anomalous EPR spectral patterns have also been seen in Glycera Hb (Hori et al., 1990) and BrCN-modified sperm whale Mb (Hori et al., 1989). All of these are five-coordinate ferric hemoproteins. This implies that five-coordinate ferric species may be a prerequisite for the quantum mechanical admixture of intermediate spin state in hemoproteins. Ligand field considerations indicate that the quantum mechanical admixture could be stabilized in a heme-

    H. Hori and M. Ikeda-Saito, unpublished results.

  • Ferric Mb Mutants 22851

    iron configuration in which the iron atom is somewhere between the out-of-plane position of typical high spin hemes and the nearly in-plane position associated with low spin hemes (Maltempo, 1974). In this context, x-ray crystallo- graphic studies indicated that the displacements of the iron atom from the mean heme plane are 0.26 8, for Aplysia Mb (Bolognesi et al., 1989) and 0.4 8, for sperm whale met Mb (Takano, 1977), respectively. The spin state of sperm whale ferric Mb H64V was assigned as high spin S = 512 on the basis of the hyperfine shift pattern of the proton resonances (Rajarathnam et al., 1991), a technique reported to be able to discriminate between S = 312 and S = 512 states in several cytochrome c’ proteins (La Mar et al., 1990). Our EPR results indicate that H64V is a mixture of approximately 95% high spin and 5% intermediate spin state at 6 K. The high spin S = 512 species predominantly exists in ordinary solutions at room temperature (Smulevich et al., 1991), and the small amount of the S = 312 state present may not be detected by ‘H NMR.

    One may claim that “the buffer effect” observed in the EPR spectra might come from freezing induced changes in the pH values. We doubt that this is the case, because the observed pH effects differ from one mutant to another; and MES and HEPES are Good buffers, the pH of which have been consid- ered not to change drastically upon freezing (Williams-Smith et al., 1975). The low spin EPR signal with gl = 2.77, g2 = 2.28, g, = 1.61 in H64V and H64Q in HEPES can be catego- rized as the “H-type” described by Blumberg and Peisach (1971) or the “N,N-type” of Yoshimura and Ozaki (1984), which is characterized by the ligation of an imino nitrogen at the sixth coordination position of the heme iron. We find it very difficult to propose a plausible origin of this low spin state in H64V and H64Q in HEPES, since HEPES does not have any imino nitrogen, and since Val cannot be a sixth ligand of the heme iron either. The g value of another low spin signal (gl = 2.53, g, = 2.17, g, = 1.86) discernible in H64Q in HEPES is the same as that of the alkaline OH form of H64Q. This may be due to a freeze-induced slight pH increase, or HEPES may shift the acid-base equilibrium of H64Q by interacting with the distal Gln. In H64V and H64L, measure- ments in phosphate or Tris buffers yielded g, signals with reduced rhombicity in comparison with those in MES or HEPES buffers. Similar buffer effect was reported by Emp- tage et al. (1977) for the EPR spectrum of R. rubrum cyto- chrome c’. Although we do not have a plausible explanation for the buffer effects, it would be advisable to carry out ferric hemeprotein EPR measurements in several buffer systems.

    The distal His + Gln replacement slightly increases (3.5 times) the azide affinity of human Mb, while replacing the distal His with Leu or Val considerably decreases (about 50 times) the azide affinities. The increased azide affinity in H64Q suggests more favorable interactions between the bound azide and the E7 residue in H64Q than in H64H. Steric hindrance due to the side chain volume of the E7 residue does not seem to play an important role in the azide affinity of metMb, as the azide affinity of H64G, which has been consid- ered to have a sterically unhindered distal heme pocket (Rohlfs, et al. 1990), is about 10 times weaker than that of H64H.5 We suspect that the polar nature of the side chain of the distal E7 residue is significant in azide binding to the ferric Mb. Similar conclusions were also reached for 0, bind- ing in ferrous Mb (Rohlfs et al., 1990). Rohlfs et al. (1990) also showed that the distal His -+ Val and + Leu replace- ments induced larger changes in the ligand affinities of ferrous sperm whale Mb than the His -+ Gln mutation. This also seems to be the case for the azide affinity of ferric Mb.

    Cutruzzoli et al. (1991) reported that the distal His + Val mutation in sperm whale ferric Mb increased azide on and off rate constants by factors of 10’ and 2.5 x lo3, respectively, resulting in a reduction of azide affinity in H64V. It is likely that both on and off rates are increased in the human Mb mutants as seen in the sperm whale counterpart, as most of the effects of mutation on human Mb are essentially parallels to those on the sperm whale protein. Under similar experi- mental conditions, the azide affinity of ferric Aplysia Mb (dissociation equilibrium constant of 400 pM (Giacometti et al., 1975)) is between those of H64H and H64V. The increased azide affinity in Aplysia Mb over H64V could be primarily due to interactions between the bound ligands and the side chain of the E10 Arg residue as reported by Bolognesi et al. (1990) and Cutruzzoli et al. (1991).

    The His + Leu substitution at the E7 site causes an approximate 50-fold reduction in the cyanide binding rate constant in human ferric Mb. Our low temperature photolysis experiments have shown that the distal heme pocket in H64H is more crowded than that in H64L (Ikeda-Saito et al., 1991). Steric constraints in a crowded distal heme pocket would retard cyanide binding, which energetically prefers linear iron ligand bonding (Fe-C-N), by mandating a tilted or bent Fe- C-N structure (Yoshikawa et al., 1985). H64L, with a less crowded distal binding site, is expected to react with cyanide faster than the wild type with its more crowded distal heme pocket. On the contrary, H64L binds cyanide more slowly than H64H. Anomalously slow cyanide binding to Glycera Hb was reported by Mintorovitch and Satterlee (1988). The pres- ence of Leu at the E7 site seems to be one of the major causes for the reduced cyanide association rate. It should be pointed out that this differs from the case in the ferrous protein: the distal His + Leu replacement increases the 0, and CO asso- ciation rates to ferrous sperm whale Mb (Rohlfs et al., 1990). One may suggest that this opposite effect on the ligand binding rate is related to the valence state of the iron, since ferric Mb binds more ionic ligands, such as cyanide and azide, while ferrous Mb binds less ionic ligands, such as O2 and CO. This suggestion, however, cannot explain the 10’ increase in the association rate constant of azide binding to sperm whale ferric Mb upon the distal His + Val mutation (Cutruzzola et al., 1991). The difference in the cyanide on rates between H64L and H64V could not be attributed to distal pocket crowding, as cyanide binds faster to H64V which has the more restricted distal pocket (Ikeda-Saito et al., 1991). A major difference between H64L and H64V is that Leu could be considered to be more hydrophobic than Val, and it might be deduced that the aliphatic nature of the distal E7 residue could be contributing to the reactivity of ferric Mb. Cutruzzola et al. (1991) attributed the faster azide binding in H64V to the vacant sixth coordination position: ligand substitution required for azide binding in water-coordinate H64H slows down the azide binding process. This notion, however, cannot explain the slower cyanide binding to H64V than to H64H. It is likely that a number of factors determine the ligand binding rates involved in ferric hemoproteins. These factors probably include the structure and hydropathy of the distal pocket, and the hydrophobicity, geometry, size, and anionic nature of ligands.

    It would be interesting to compare the properties of the ferric mutants with those of natural distal His variant he- moproteins and the ferrous mutants. Upon comparison of the kinetic parameters of ligand binding to the ferrous sperm whale Mb distal His mutants with those of Glycera Hb, Aplysia Mb, and elephant Mb, Rohlfs et al. (1990) concluded that (i) H64L has functional characteristics similar to Glycera Hb,

  • 22852 Ferric M

    (ii) H64Q resembled qualitatively but not quantitatively ele- phant Mb; but (iii) there is little correlation between the functional behavior of H64V and that of Aplysiu Mb. Our results indicated that although both ferric Glyceru Hb and H64L are five-coordinate species, they are very different. Ferric H64L binds cyanide 10 times faster than Glyceru ferric Mb. The electronic structure of the heme iron in H64L is different from that of Glyceru Hb as judged by the present EPR results. Structural differences other than the E7 position in Glyceru Hb (Arents and Love, 1989) must be responsible for these differences, and H64L is not necessarily a good model for Glyceru Hb. The EPR spectral properties of ferric H64V are different from those of Aplysia Mb. Thus, in agree- ment with the results of Rohlfs et ul. (1990), we think that the distinctive features of Aplysiu Mb are due not only to the distal His -+ Val replacement, but also to other key heme pocket amino acids such as Asp45 and ThF7 (Bolognesi et ul., 1990). Lack of EPR and ligand binding data on elephant ferric Mb precludes assessment of the electronic structure of its heme iron with reference to that of H64Q from the present study.

    Acknowkdgrnents-We thank Dr. S. G. Boxer and S. Balasubra- manian for the human Mb expression system and suggestions, Dr. G. N. Phillips, Jr. for the atomic coordinates of the sperm whale Mb mutants, Dr. S. Kimura for oligonucleotides synthesis, Dr. J. H. Dawson for a preprint of their MCD paper, and L. L. Monette for assistance.

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