0 1984 by The American Society of Biological Chemists, Inc. THE JOURNAL OF BIOLOGICAL CHEMISTRY Val. 259, No. 4, Issue of February 25, pp. 2092-2099, 1984

Printed in U.S.A.

Catalysis of Nitrosyl Transfer Reactions by a Dissimilatory Nitrite Reductase (Cytochrome c,dl)*

(Received for publication, August 31, 1983)

Choong-Hyun Kim and Thomas C. HollocherS From the Biophysics Program and Department of Biochemistry, Brandeis Uniuersity, Walthum, Massachusetts 02254

The dissimilatory nitrite reductase (cytochrome c,dl) from Pseudomonas aeruginosa was observed at pH 7.5 to catalyze nitrosyl transfer (nitrosation) between [I6N]nitrite and several N-nucleophiles or H2180, with rate enhancement of the order of lo8 relative to anal- ogous chemical reactions. The reducing system (ascor- bate, N,N,N',N'-tetramethylphenylenediamine) could reduce nitrite (but not NO) enzymatically and had es- sentially no direct chemical reactivity toward nitrite or NO. The N-nitrosations showed saturation kinetics with respect to the nucleophile and, while exhibiting V,,, values which varied by about 40-fold, neverthe- less showed little or no dependence of V,, on nucleo- phile pK,. The N-nitrosations and NO;/H20-'80 ex- change required the reducing system, whereas NO/ HzO-"O exchange was inhibited by the reducing sys- tem. NO was not detected to serve as a nitrosyl donor to N-nucleophiles. These and other kinetic observa- tions suggest that the enzymatic nitrosyl donor is an enzyme-bound species derived from reduced enzyme and one molecule of nitrite, possibly a heme-nitrosyl compound (E-Fe". NO+) for which there is precedence. Nitrosyl transfer to N-nucleophiles may occur within a ternary complex of enzyme, nitrite, and nucleophile. Catalysis of nitrosyl transfer by nitrite reductase rep- resents a new class of enzymatic reactions and may present another example of electrophilic catalysis by a metal center. The nitrosyl donor trapped by these re- actions is believed to represent an intermediate in the reduction of nitrite by cytochrome c,dl.

Catalysis of nitrosyl transfer from nitrite to azide, hydrox- ylamine, and water by denitrifying bacteria was detected during active denitrification of nitrite (l), and respiratory nitrite reductase was implicated indirectly as the catalyst. Catalysis of nitrosyl transfer represents a new biochemical reaction which may provide insight on the activation of nitrite for reduction. A heme-containing purified respiratory nitrite reductase is shown herein to catalyze nitrosation reactions in vitro. Some such reactions are characterized and their bearing on the mechanism of nitrite reduction is discussed.

* This investigation was supported by Grants PCM 79-12566 and PCM 82-18000 from the National Science Foundation and Biomedical Research Support Grant SO7 RR07044 from the National Institutes of Health. The gas chromatograph/mass spectrometer was purchased under a grant from the Edith Blum Foundation to E. Grunwald, Department of Chemistry, Brandeis University. The costs of publi- cation of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertise- ment" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed.

EXPERIMENTAL PROCEDURES Materials-['5N]NaN02 (99 atom %) was obtained from Stohler

Isotope Chemical Co. (Waltham, MA). Its chemical purity was deter- mined to be >95% by colorimetric assay (2), and its I5N isotopic abundance was confirmed by GC/MS' analysis of nitric oxide pro- duced by nitrite reductase purified from Pseudomonas aerugimsa (3). ['BO]HzO (97 atom %) was obtained from KOR Isotopes (Cambridge, MA). Its isotopic purity was confirmed by MS analysis as reported previously (1). Commercial (tank) NO was purchased from Matheson Co. (Gloucester, MA), and contained typically 0.6 mol % of N20 as determined by GC/MS analysis and about 0.5 mol % of NO, as determined by the supplier. NO was passed through a NaOH pellet column to remove NOz. I6NO was chemically synthesized from [15N] NaN02 using FeS04 and NaBr as reductants (4) and was essentially free of "N20 by GC/MS analysis.

Stock solutions of sodium ascorbate (1 M, containing 10 mM disodium EDTA) and TMPD (30 mM) were prepared in 100 mM potassium phosphate buffer, pH 7.5. Aniline was redistilled and stored under Nz. Stock solutions (1 M) of hydroxylamine, p-methoxyaniline, hydrazine, trifluoroethylamine, and ethylenediamine were prepared from their respective hydrochloride salts which had been recrystal- lized. All the stock solutions were prepared and titrated to pH 7.5 immediately before use and were maintained thereafter under anaer- obic conditions.

Bacterium-P. aerugimsa PA01 was obtained from B. W. Hollo- way, Monash University, Melbourne, Australia. Stock cultures were maintained on nutrient agar slants and plates. Bacteria from a single colony were passed through three successive cycles of growth at 30 "C. The first cycle was aerobic growth in 50 ml of medium containing 3 g of beef extract and 5 g of bacto-peptone/liter of water; the second (500 ml) and third (50 liter) were anaerobic growth in the same medium supplemented with 10 mM potassium nitrate (2). Cells from the third cycle were harvested by continuous flow centrifugation about 20 h after incubation (3).

Purification of Nitrite Reductase-The dissimilatory nitrite reduc- tase (cytochrome c,dl; Pseudomonas cytochrome oxidase; ferrocyto- chrome c-551:oxidoreductase; EC 1.9.3.2) was purified from P. aeru- ginosa by the method of Parr et al. (5), with a modification suggested by C. Greenwood (3). The enzyme was stored at -20 "C for several months without much loss of activity.

Gas Chromutographic/Mass Spectrometric Technique-The amount and isotopic content of Nz, NO, and N20 in the gas phase of the reaction systems were determined by means of a Hewlett-Packard 59926 GC/MS equipped with a Porapak Q column (2.4 m X 3.2 mm) operating at 38 "C, as described previously (1, 3, 6). Nz was analyzed for the "N-labeled species at m/e values of 29 and 30. The amount of I4N2 (m/e = 28) produced was sometimes not determined due to interference by the air background. Absolute amounts of NO, N20, and N2 were determined with reference to external standards. Relative amounts of these gases were determined with reference to an internal argon standard. For most experiments, gas samples of 0.2-ml volume were collected by means of a pair of gas-tight holding valves (Uni- metrics 2375) and then expanded into the lead line/GC sample loop. However, for the analysis of small amounts of N20 or '5N-labeled N2, a sampling method was used which involved direct expansion of the head space of the sample vial into the lead line/GC sample loop (3). This method removed significant amounts of gas from the vial and

The abbreviations used are: GC, gas chromatography; MS, mass spectrometer; TMPD, N,N,N',N'-tetramethylphenylenediamine; SDS, sodium dodecyl sulfate.

2092

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Catalysis of Nitrosyl Transfer 2093

SO required corrections of the data to account for this removal. The algebraic form of these corrections is defined in Appendix A?

Reduction and Nitrosation Systems-Reaction mixtures were typ- ically 1 ml in volume, and were contained in 9-ml vials, each with a magnetic stirring bar. Normally, the solvent was 100 mM potassium phosphate buffer, pH 7.5, but when H;'O was used, the concentration of potassium phosphate buffer was 70 mM. The vials containing reaction mixture were sealed by means of a serum bottle stopper and made anaerobic by repeated evacuation and back filling with helium. Final helium pressure was up to 1.5 atm. 0.1 ml of argon at 1 atm was added to each vial as the internal MS standard. To the vials was then added one or more of the following components (each as an anaerobic stock solution): 0.5 nmol of enzyme, 0.6 pmol of TMPD, 40 pmol of ascorbate with 0.4 pmol of EDTA, and some quantity of a nitrogen nucleophile, in that order. Additions were made by means of gas-tight syringes. Reactions were started by injection of NO (2-4 pmol) and/ or nitrite (10-40 pmol). The reactions (nitrite reduction and nitro- sation of nucleophile) proceeded at 20 -+ 1 "C with vigorous stirring in order to rapidly equilibrate the gas and aqueous phases (2). Aliquots ofthe aqueousphase (0.1 ml) were sampled by use of gas-tight syringes purged with helium. The amount and isotopic content of nitrite in these aqueous samples were determined by analysis.

Assays-Protein was determined by absorption at 280 nm (5) and by the method of Lowry et al. (7), and nitrite was assayed colorimet- rically (8).

Isotopic analysis of nitrite was carried out by first converting nitrite to N20 by reaction with azide under acidic conditions (1). The N20 so produced was then analyzed by GC/MS for 15N and "0.

Nitrite reductase was assayed with the ascorbate/TMPD-oxidase reaction at 30 'C (3, 5). The oxygen electrode was a Clark oxygen electrode, Yellow Springs Instrument Co., Model 53.

Optical spectra were obtained by use of a Perkin-Elmer Model 559 recording spectrophotometer.

The procedure of Weber and Osborn (9) was followed by SDS-disc electrophoresis (4 h at 150 V), but the polyacrylamide gel and protein samples were prepared by Laemmli's method (10). The gels were stained with Coomassie blue prepared in a mixture of ethanol, acetic acid, and water (5:1:5) (v/v) and destained in a mixture of these solvents in a ratio of 1:1:9. The stained bands were scanned at 570 nm by means of a densitometer consisting of a Gilford Model 222 spectrophotometer and a Gilford Model 6X2D linear transporter which moved a quartz chamber holding the disc gel perpendicularly to the light beam.

RESULTS

Purification of Nitrite Reductase-The preparations were dark green in color and exhibited a specific activity of about 5 pmol of O2 consumed x min" X mg of protein". Although this value was 3 to 4 times that reported by Parr et al. ( 5 ) , the increases in specific activity of about 24-fold between the supernatant from the sonicated cell suspension and the final preparation were nevertheless similar to the increase reported by those workers. The total activity recovered was about 11% that in the supernatant from the sonicated cell suspension. Oxidized and dithionite reduced spectra of the final prepara- tion resembled closely those reported previously for cyto- chrome c,dl from P. aeruginosa (11-14) and four other deni- trifying bacteria (15-18). A&/AZ& ratios ranged between 1.10 and 1.29 and imply purities between 92 and 100% based on a reported value of 1.2 for the absorbance ratio of pure cyto- chrome c,dl (14). Purities of >90% were implied from densi- tometry of stained gels following SDS disc electrophoresis (19). This method also indicated that the enzyme was free of azurin and cytochrome c-551.

Portions of this paper (including Appendices A, B, and C , and Scheme C1) are presented in miniprint at the end of the paper. Miniprint i s easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Doc- ument NO. 83 "2523, cite the authors, and include a check or money order for $2.40 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

Catalysis of Reduction and Nitrosation Reactions-Reduc- tion of ["Nlnitrite by the nitrite reductase/ascorbate/TMPD system produces 15N0 and smaller amounts of 15N,0 (3). However, catalysis of nitrosyl transfer from ["Nlnitrite to azide by nitrite reductase would yield l4NI5NO (detected as 14p15N20) which is easily distinguishable from 15N20 (1). Sim- ilarly, nitrosyl transfer to aniline or primary alkylamines would yield 14.15N2, which is also a distinctive product. NP arises from solvation of diazonium precursors in these well known reactions.

The production of 14.15N20 from azide and ["Nlnitrite was readily detected and Table I summarizes the requirements for this and the reductive reactions. Nitrosyl transfer to azide required both nitrite reductase and the reducing system, as did the reductive reactions. The reducing system was unable alone to reduce nitrite or NO chemically at detectable rates or to activate nitrite for nitrosyl transfer at pH 7.5. Nonen- zymatic nitrosation of azide by nitrite (actually by nitrous acid) was extremely slow at pH 6.0 but not detectable at pH 7.5. Ascorbate alone (Table I) could serve as an inefficient reductant for the enzymatically catalyzed reduction of nitrite, the rate of reduction being at least 15 times smaller than that observed with ascorbate plus TMPD. A corresponding de- crease in the rates of production of 15N20 and 14.15N20 would have made such reactions effectively undetectable. I5NO was neither reduced to 15N20 nor competent as a nitrosyl donor in the enzymatic reactions. The results of Table I are also representative of the several N-nucleophiles used, in addition to azide. EDTA was used to chelate traces of free iron, which can catalyze certain reactions, such as the nonenzymatic reduction by NO by ascorbate (20).

Both the enzymatic reduction of ["N]nitrite and nitrosa- tion of azide showed rates that decrease with time (Fig. I), probably as the result of progressive inhibition by NO (14,21, 22), the chief product of the reductive reaction. The initial rates of production of I5NO, I5NZO, and 14J5N20 in this exper- iment were 636,19, and 50 pmol x min" X pmol of enzyme-', respectively. Note that 50 mM azide had little effect on the reductive reactions and that the presence of azide was required for production of 14,15N20.

TABLE I Characterization of reaction products in the presence of NaNa

System Reduction products N ~ ~ ' ~ ~ ~

"NO "N20 11.16N20

Nitrite as source of I5N

Complete" +++ ++ +t Minus enzyme - - Minus TMPD ++ - - Minus ascorbate, - - -

-

EDTA, TMPD

Nitric oxide as source of 15N

Complete Minus enzyme Minus TMPD, ascor-

bate,EDTA +b

Minus enzyme, TMPD, - +b

ascorbate, EDTA The complete system contained 0.5 nmol of enzyme, 20 pmol of

[16N]nitrite (or 4 pmol of NO), 40 pmol of ascorbate, 0.4 pmol of EDTA, 0.6 pmol of TMPD, and 50 *mol of NaN3 in 1 ml of 100 mM potassium phosphate buffer, pH 7.5. Typically, +++, ++, +, and - stood in the mole ratio 100:5:1.5 to 0.5:<0.5.

bAn artifact caused by traces of 0 2 . NO and O2 form N20a tran- siently which nitrosylates azide chemically. Typically, the amount of 14,15N20 produced by this process was ~ 0 . 2 pmol.

- - - - -

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2094 Catalysis of Nitrosyl Transfer

15 t . I

1 I I J ~

0 30 60 90 T m e , Mln

FIG. 1. Kinetics of gaseous N-oxide production from ['"N] nitrite in the absence and presence of azide. The reaction system was the same as the complete system containing ["NJnitrite described in Table I. The reaction was initiated by addition of [''Nlnitrite. Note that the vertical ais is scaled differently for "NO and the nitrous oxide species. 0 and 0, 15NO; A and A, 14N''NO; 0 and H, 15Nz0. Open symbols, absence of azide; closed symbols, presence of 50 mM azide. The systems minus enzyme and/or minus reducing system performed as per the open triangles of this figure.

1 I

P e a

0 IO 20 30 4 0 5 0

[NONS] I mM

FIG. 2. Dependence of nitrosyl transfer to azide on the con- centration of azide. The reaction system was the same as the complete system containing ["Nlnitrite described in Table I, except for varying azide concentrations. The product mole ratio (vertical axis) is based on the results of analysis performed 15 min after initiating the reaction. 15N0 and 15N20 are the major and the minor products of reduction of nitrite, respectively, and "N''NO is the product of nitrosyl transfer to azide. 0, the 14N1'N0f5N0 ratio; A, the '5N,0/'6N0 ratio.

If one considers initial rate data, the enzymatic nitrosation of azide, typical of the N-nucleophiles studied, showed satu- ration kinetics (Fig. 2). The initial rates of the nitrosation reactions became zero order in the concentration of the nu- cleophile by or about 50 mM, and K, values ranged between about 2 and 20 mM among those nucleophiles examined. K, for azide (11 mM) and aniline (4 mM) were quantitated; those for the other nucleophiles were estimated qualitatively.

Summarized in Table I1 are reduction and nitrosation prod- uct yields (based on initial rate data) for several N-nucleo- philes which were chosen to provide a variety of pK, values and chemical types and to yield NzO and/or N2 as the product of nitrosyl transfer. Under the conditions employed, the rates of breakdown of the primary nitrosation products (e.g. O15NN3 in the reaction of azide and ["Nlnitrite) to final products were apparently never rate determining. No lag in formation of 14,15N20 or I4,l5N2 was observed, as could occur if breakdown of a nitrosylated intermediate were slow. Rate limiting break- down would be expected to be first order (not zero order) in

the nucleophile and, in addition, alkyl and aryl diazonium intermediates are expected to hydrolyze rapidly at pH 7.5 (23-26). The data of Table I1 refer to nitrosation V,,, (also reduction V-) except for the nitrosation of hydrazine and ammonia which were not present a t saturating concentra- tions. The observed rates for nitrosation of ammonia and hydrazine in Table 11, were about 70 and 50% of Vmax, respec- tively. In general, the nucleophiles at the concentrations used had little effect on the rates of the reductive reactions. At the bottom of Table I1 is illustrated the observation that when two N-nucleophiles are present at equal concentrations, the one with the lower K,,, (here aniline) inhibited nitrosation of the one with the higher K, (here azide), but the obverse inhibition was not prominent.

The nitrosation rate data of Table I1 are plotted logarithm- ically (after background and statistical corrections) against the statistically corrected pK, of the nucleophiles (Fig. 3). The rate value for the hydrazine point was determined from Table I1 as outlined in Appendix B. While the several nucleo- philes exhibited corrected nitrosation V,,, values that vary by a factor of about 40, there seemed to be little overall dependency of this parameter on pK,. Thus, the apparent value for Pnue is zero in the enzyme catalyzed nitrosation of N-nucleophiles.

Catalysis of nitrosyl transfer to azide was measured at 10, 20, and 40 mM [l'NJnitrite (Table III), but changes in the initial rates of formation of products or in the '5NzO/'5N0 and 14,15N20/15N0 product mole ratios were not observed.

Catalysis of Oxygen Exchange-A consequence of catalysis of nitrosyl transfer to general nucleophiles should be catalysis of oxygen exchange between nitrite and H,"0. If enzyme- bound nitrite can undergo oxygen exchange and then experi- ence reduction instead of dissociation as [180]nitrite, then the NO or NzO so produced must also be labeled at the outset before the nitrite pool becomes significantly labeled (1). In fact, the enrichment of in NO and NzO relative to that in the nitrite pool is a measure of the "stickiness" of the enzyme with respect to nitrite and of the mean number of oxygen exchange events occurring during the lifetime of the enzyme- nitrite complex (1).

When reduction of [15N]nitrite by nitrite reductase was carried out in HZ"O (Fig. 4), it was observed that newly produced l5N2O and 15N0 were always more highly enriched in I8O than was the nitrite pool and that the "0 enrichments of 15N20 and 15N0 were very similar. Without the reducing system, there was no measurable reduction of nitrite and little incorporation of into the nitrite pool. When 50 mM azide was included in the complete system of Fig. 4, it was found that I5N2O, 15N0, and 14,15Nz0 (the latter from the nitrosation reaction) all had similar "0 enrichments. In one experiment, the (cumulative) l80 content of these species was 4.3,3.5, and 3.9 atom %, respectively, after an incubation time of 15 min in 30 atom % H280. These observations require that reduction and nitrosation reactions all stem from a common enzyme- bound precursor which is more highly enriched in "0 than is the nitrite pool.

A kinetic model for l80 exchange is developed in Appendix C. The model suggests that acquisition of "0 in enzyme- bound nitrite and its reduction proceed at similar rates, but that once [180]nitrite is formed on the enzyme, its dissociation is more probable than is its reduction. The enzyme is thus mildly "sticky" with a rate constant for nitrite dissociation being somewhat greater than that for reduction. The most probable number of exchange events before reduction of enzyme-bound nitrite is approximately 0.1.

The oxygen exchange reactions would appear to be consist-

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Catalysis of Nitrosyl Transfer 2095

The pK. values are from Ref. 60. Total concen- tration of base and conjugate pK,

acid Nucleophile

mM

None added NB 50 4.72 NHzOH 50 5.98 H2N-NH: 50 7.99 CsHS-NHz 50 4.58 p-CH30-CsHS-NH2 50 5.29 FaCCHz-HNZ 50 5.70 HzN-CH2CHZ-NHj 100 7.52 NH, 1000 9.24 N: plus 50

CsHS-NHz 50 Like first row. 0.0014 f 0.0003.

Concentra- Initial rate Product mole ratio tion of free of lsNo pro- base at pH duction

"Nz0 - 14.16NNz0 __ 7.5 "NO "NO "NO - 14.16N

min" X pmol X

pmol en- zyme"

mM

636 0.030 0.0005 0.0014 49.9 588 0.032 0.079 48.5 936 0.030 0.057 12.2 614 0.032 0.010 49.9

0.0029 420 0.029

49.7 475 0.025 0.017 0.012

49.2 610 0.025 0.0033 48.8 541 0.036 0.0035 17.9 421 0.037 49.9

0.014

0

c

49.9} 437 0.028 0.048 0.014

TABLE I1 Product ratios for reductive and nitrosation reactions in the presence of various nucleophiles

The reaction system was the same as the complete system containing [15N]nitrite described in Table I, but various amounts of other N-nucleophiles replaced azide. The average rate for the initial 15-min period is taken to approximate the initial rate. 14915N20 and "*15N2 are nitrosation products; 16N20 and "NO are reduction products.

I

-

See Appendix B for an evaluation of the overall rate of nitrosation of hydrazine. ~~

Like first row, 0.0005 0.0002.

0

0 0, - I

O t

I I 0

I - O 4 5 6 7 8 9 IO 15 16

PKo + log P/q

FIG. 3. Relative rate of nitrosyl transfer as a function of nucleophile pK, The vertical and horizontal axes in the log-log plot represent the normalized initial rate of nitrosation (or "0 exchange) and the pK, of the nucleophiles, respectively. Both parameters have been statistically corrected (27,61,62). The rate ratios represent V,, values except for NH2NH2 and NH3. The tip o f the arrows for NH2NH2 and NH3 indicate the expected values a t V,, (see text and Appendix B). The statistical correction factors are as follows: p is the number of equivalent dissociable protons on the conjugate acid and q is the number of equivalent points at which a proton can become attached on the conjugated base.

ent with Equation 1, in which enzyme-bound nitrosyl, NO', is the common precursor in reduction and nitrosation reac- tions.

It also ought to be possible in principle to form enzyme-bound nitrosyl through Equation 2 , i.e. from oxidized enzyme and NO, and thereby to detect a NO/H20-'s0 exchange reaction.

E-Fe"' + NO e E-Fe"'.NO e E-Fe".NO+ (2)

A E-Fe" .NO, + 2H+ kHz0

That reaction should be inhibited by the reducing system just as the NO;/HzO-'sO exchange (Fig. 4) was prevented by lack

TABLE I11 Products from reduction of P5N]nitrite and nitrosation of azide at

different concentrations of nitrite The reaction system was the same as the complete system described

in Table I, except with regard to nitrite concentrations. 14*16N20 is the nitrosation product; I5N2O and 15N0 are reduction products.

pmoI X min"

zyme-' mM X pmol en-

10 557 0.026 20

0.078 617 0.03 1 0.070

40 516 0.032 0.084 ~ ~~

of the reducing system. A reducing system inhibited NO/H20- '"0 exchange was in fact detected (Fig. 5). The observation of I8O exchange with both nitrite and NO under appropriate conditions suggested that all steps of equation 3 are in prin- ciple easily reversible.

E-Fe" + NO; e E-Fe".NO; E-Fe".NO+ &2H+

&HZ0 + E-Fe"'. NO e E-Fe"' + NO (3 )

Attempted Detection of a N0Z/NO-l5N Exchange-At- tempts to demonstrate the reversibility of Equation 3 directly by means of a NO;/NO-"N exchange reaction were unsuc- cessful or inconclusive. In presenee of the ascorbate/TMPD reducing system, reversibility could not be expected because a reductive reaction, probably Equation 4, would serve to make Equation 3 effectively irreversible from left to right.

lh Ascorbate + E-Fe"' - 112 Dehydroascorbate + E-Fe" (4)

Failure to observe the 15N exchange in absence of the reducing system (with 20 pmol of [I5N]nitrite plus 4 @mol of NO) can be attributed to at least two factors, which are considered below.

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Catalysis of Nitrosyl Transfer

0 30 60 90 Time, Min.

FIG. 4. Incorporation of "0 from H2"0 into the reduction products of nitrite and the nitrite pool. The system contained 0.5 nmol of enzyme, 20 pmol of ["Nlnitrite, 40 pmol of ascorbate, 0.4 pmol of EDTA, and 0.6 pmol of TMPD in 1 ml of 70 mM potassium phosphate buffer, pH 7.5. The sovent water contained 30 atom % H2180. 0, I5NO; A, 15Nz0; 0, [15N]nitrite; 0, [15N]nitrite in absence of reducing system and EDTA ., [I5N]nitrite in absence of enzyme. Data by intervals provides a measure of the "0 content of I5NO and 15N20 newly produced during each 5- or 10-min interval. Cumulative data provides a measure of the "0 content of the [I5N]nitrite pool at each point in time.

m - I I

0 30 60 90 Tlmc. Min.

FIG. 5. Incorporation of "0 from H2"O into the "NO pool under different conditions. The complete reaction system was the same as that for Fig. 4, except that 15N0 (3.6 pmol) replaced ["N] nitrite. 0, complete system; A, minus the reducing system and EDTA; 0, minus the reducing system, EDTA, and the enzyme.

DISCUSSION

In this study, catalysis of nitrosyl transfer from nitrite to N-nucleophiles and water was demonstrated with a purified dissimilatory nitrite reductase of the cytochrome c,dl type from P. aeruginosa. The characteristics of the reactions sug- gest Scheme 1 as a model. Central to both reduction and nitrosation reactions is a nitrosyl donor, designated as E-Fe"- NO+, formed by dehydration of nitrite. This species can be viewed as the activated form of nitrite and the immediate precursor of both reduction and nitrosation products. The NO;/H20-180 exchange (reducing system requiring) and the

NO/HZO-'~O exchange (reducing system inhibited) suggest that reactions between nitrite and NO are in principle revers- ible. Recent isotope studies (3) require that NO and NzO production be represented as parallel (not sequential) path- ways for this particular enzyme and reducing system. That nitrosation of N-nucleophiles showed saturation kinetics but different Vmax values is consistent with formation of a ternary complex. The interference by one nucleophile of the nitrosa- tion of another with the higher K,,, is also consistent with involvement of a ternary complex. The reduction of enzyme by ascorbate-TMPD is represented as an essentially irrevers- ible reaction in order to rationalize the failure to observe a NOT/NO-l5N exchange in presence of the reducing system. The reactions are depicted as involving nitrite, but it is possible and indeed likely that the reactive species is HN02, because in principle nitrite lacks a suitable leaving group for dehydration (27-29). There is, in addition, evidence that HNOZ is the true substrate in the reduction of nitrite to NO by hemoglobin (30).

Model for the Nitrosyl Donor-Chemical nitrosations by HNOz in aqueous solution proceed by way of Nz03 and/or NO+ (or HzNO:)~ (23, 28, 29, 31). Free nitrosyl donors such as these can be ruled out in the enzymatic reaction by at least two arguments. First, although the enzyme can be imagined to catalyze the dehydration of HN02, it must also catalyze the reverse reaction and so could not be expected to affect the equilibrium among nitrite, HN02, and the above free nitrosyl donors. The equilibrium concentrations of these donors is insufficient at pH 7.5 to bring about measurable rates of nitrosation or 0 exchange. Second, the enzymatic nitrosyl donor has reactivities rather different from those of strong chemical nitrosyl donors such as NO+, Nz03, or NOCl. Reac- tions of N203 generated from NO plus NO, with anilines and secondary alkylamines are first order in the nucleophile and show no dependency of rate constant on nucleophile pK. above a pK, of about 0 (25). NOCl behaves similarly (26), and NO+, which is considered to be the strongest nitrosyl donor, is also relatively unselective toward N-nucleophiles (29, 31, 37,38). N203 generated from HN02, a weaker nitrosyl donor which is thought to be largely ONNO* rather than ONON0(25), prefers to react with more strongly basic N- nucleophiles (29, 31, 38) and shows Pnue of 0.8 to 1.0 with alkylamines in the pK, range 9-11 (31). By comparison, the nitrite reductase system showed saturation kinetics, but, like the strongest chemical donors, showed no dependence of rate (Vmax) on pK,. N-nucleophiles are about 1000 times more reactive than is water (at the same concentration) in nitrosyl transfer from N ~ 0 3 (generated from NO plus NO,) and NOCl (24-26). If one assumes that the rate of N0i/H20-180 ex- change is proportional to water concentration in the enzyme system, then the reactivity of azide (the most reactive N- nucleophile studied) was only about 125 times that of water. If the 0 exchange reaction were also to show saturation kinetics with respect to water, then water must be an even more reactive nucleophile relative to azide and, in the limit, could be about 8 times more reactive than azide (Fig. 3). We conclude, therefore, that catalysis of nitrosation must proceed

The involvement of NO+ as opposed to HzNO; in acid catalyzed nitrosation reactions remains a matter of controvery (29). Nitrous acid acidium ion is suggested by the slow HN02/HzO-"O exchange relative to nitrosation (32) and by failure often to observe a change from a first to a zero order dependency on nucleophile concentration at high concentrations of the nucleophile (29, 33, 34). The nitroson- ium ion is suggested by kinetic studies in which oxidation of nitrous acid by H2O2 competes with nitrosyl transfer (35, 36). The weight of evidence seems to favor involvement of NO+ rather than HZNOZ (29) in these reactions.

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Catalysis of Nitrosyl Transfer 2097

X:

- E-FeI" e, E-Fe"

SCHEME 1

by way of an enzyme-bound nitrosyl donor formed between nitrite (or HN02) and reduced enzyme. This species is repre- sented in Scheme 1 as a ferrous heme nitrosyl compound (1, 39) for which chemical precedence exists. Metal stabilized NO+ includes nitroprusside anion, [(CN)5FeN0]2- (40), the nitrosyl of iron tetraphenylporphyrin chloride (41), and nu- merous ruthenium, iridium, and osmium nitrosyl compounds (42,43). Metal nitrosyl compounds such as these can serve as nitrosyl donors variously to N-, 0-, S-, and C-nucleophiles and some can undergo oxygen exchange. Metal nitrosyl com- pounds are generally formed by reaction of NO with an oxidized form of the metal (40-43). The formation of hemo- protein iron-nitrosyl from NO and ferric hemoproteins has also been reported (44-47). The resulting compound in the case of cytochrome c has been reported to be diamagnetic (44) as expected for a low spin Fe".NO+ compound. Spectropho- tometric evidence for formation of Fe". NO+ species by reac- tion of oxidized cytochrome c,dl with NO also exists (14, 21).

The present data cannot entirely rule out the possibility that the true nitrosyl donor is enzyme-bound N203, formed by reaction of nitrite as a nucleophile with E-Fe". NO+. Such a species (ONN02) has been postulated to represent an inter- mediate in a hypothetical scheme for the reduction of nitrite by denitrifying bacteria (39). Two points of evidence support the view that the enzymatic nitrosyl donor is monomeric with respect to nitrite and therefore is not bound Nz03. First, the l80 content of the reduction products and the NzO formed by nitrosation of azide were the same. The "0 content of the latter product was not strongly diluted with unlabeled oxygen from nitrite, as would be expected to occur if the nitrosyl donor were dimeric (N203) with respect to nitrite. This obser- vation is the same as that made previously with intact deni- trifying bacteria (1). Second, the product ratios at V,, among the two reduction products and the nitrosation product were not influenced by the nitrite concentration (Table 111). Thus, at each partition point in Scheme 1, the order of the reactions with respect to nitrite was the same. Because one partitioned reaction leads to a monomeric product (NO), it is likely that all three partitioned reactions issue from precursors mono- meric with respect to nitrite. This observation also supports other lines of evidence (1, 48, 49) to the effect that N20 is formed in denitrifying bacteria by dehydrative dimerization of nitroxyl (NO-).

Formation of E-Fe".NO+ can be imagined to experience general acid catalysis or electrophilic catalysis by an adjacent heme, as in Scheme 2.

Catalytic Rate Enhancements-The extent of catalysis can be estimated by comparing an appropriate rate constant of

n 0

SCHEME 2

the enzymatic reaction with an analogous one of the same order from a corresponding chemical reaction (27, 50). For the NO;/Hz0-180 exchange, kcat of the enzymatic reaction was 6.08 s" at pH 7.5 (Appendix C). The chemical pseudo- first order rate constant, kl, for N02/H20-'s0 exchange was reported to be 7.8 X 1O" j s" at pH 6.264 with the rate equation being rate = k2[H+][HN02] = k'[H+]'[NO;] (51). Thus, kl = k'[H+]' = 2.58 x ~ O - ' S - ~ at pH 7.5. The ratio of k,.,/kl at pH 7.5 is about 2.4 X 10'.

For nitrosation of aniline by nitrite, kcat/(Km X Km') = 5.8 X lolo M-' s-' for the enzymatic reaction at pH 7.5 from kcat = 0.077 s" (Table II), K,,, (for HNOJ = 3.34 X 10"" M (as calculated from pK, = 3.3 for HNO,, K,,, = 5.3 X 1O"j M for nitrite (52)) and Kk (for aniline) = 4 X M. For the acid- catalyzed nitrosation of o-chloroaniline (rate = ks[H+] [HN02] [ClPhNH,]) the third order rate constant is reported to be 175 M-' s" (37, 53). A comparison of these two third order rate constants indicates a 3.3 x 108-fold rate enhancement by the enzyme. It is expected, due to low nucleophile selectivity, that the third order rate constant should be the same between o-chloroaniline (pK, = 2.65) and aniline (pK, = 4.58) (25,37), although a different mechanism second order in HN02 con- centration (rate = k2[HNO2I2), implying Nz03 as the nitrosyl donor, often dominates with higher pK, anilines, including aniline itself (31,33). A similar comparison of third order rate constants can be made for the nitrosation of azide anion (54), for which a rate enhancement of 8.4 X lo7 is obtained. Values of the order of 10' are not unreasonable, inasmuch as enzy- matic rate enhancements as great as 1014 are known (50, 55).

Reduction of nitrite involves inserting one or more reducing equivalents into an electron-rich species and loss of water from a species without a good 0-leaving group. It is likely therefore that a catalyst of nitrite reduction would diminish electron density and increase leaving group pK of nitrite. This by definition would be electrophilic catalysis. An analogous example is acid catalysis of nitrosyl transfer from HNO,, in which the primary nitrosyl donor in aqueous solution is NO+ (or HZNO:). Thus, it is not surprising that a nitrite reductase

'This value agrees well with the corresponding rate constant calculated from the data of Bunton and Stedman (32) when the latter were corrected from 0 "C to room temperature.

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2098 Catalysis of Nitrosyl Transfer

may also be a nitrite-nitrosyl transferase. The nitrite reduc- tase analog of NO' may be M.NO+; that of H2NO; may be

H I

M . . . 0-NO', where M represents a redox metal center. We favor participation of the former in the nitrite reductase reaction, in part because we are unaware of precedence for nitrosyl transfer from metal coordinated HN02.

Saturation Kinetics in Nitrosation of N-Nucleophiles-In- asmuch as cytochrome c,dl dimer contains two c and two d hemes, which may be clustered in the molecule (56), it is possible that N-nucleophiles could bind as ligands to a heme which is proximal to the nitrosated heme (57). It is difficult to imagine another type of interaction that could allow bind- ing with K,,, in the millimolar range for the variety of N- nucleophiles studied. Although N-nucleophiles could perhaps coordinate with heme iron, they can be expected in so doing to experience a decrease in nucleophilicity to the extent that the metal center may resemble a proton. Thus, the effective pK, of coordinated nucleophiles could be considerably lower than those of the free nucleophiles. Vmax could be diminished if the effective pK, were low enough, by analogy with certain chemical nitrosations of N-nucleophiles (25, 26, 31). It is possible, therefore, that the differences in V,,, values arose in part from the liganding of the nucleophiles to heme.

Variability in nitrosation Vm,, suggests that the rate-limit- ing step was not a prior step (e.g. dehydration of nitrite) common to all nucleophiles. While the actual rate-limiting step for nitrosation at Vmax is not clearly established, we tentatively assign it to nitrosyl transfer within a ternary complex.

Negatiue Results-Nitrosyl transfer from NO to a N-nu- cleophile in absence of the reducing system was not expected and was not observed (Table I). While it may be possible to generate the presumed nitrosyl donor, E-Fe".NO+, from E- Fe"' plus NO (14, 21), the subsequent irreversible transfer of the nitrosyl group to a N-nucleophile would trap the enzyme in the reduced state. Thus, nitrosyl transfer from NO may be expected only to the extent of one turnover. The present experiments were not sufficiently sensitive to make such a measurement. Nitrosyl transfer from NO to water is meas- urable, inasmuch as that reaction is reversible.

Failure to detect an enzymatic NO;/NO-15N exchange in absence of the reducing system is attributed to one or both of the following. First, the redox poise at equilibrium among nitrite, NO, E-Fe", and E-Fe"' in systems lacking reducing system may have been such as to bring one or the other form of the enzyme to a very small concentration. Thus, the forward and reverse exchange rates at equilibration could have been small by mass action effects. Second, exogenous NO is known to inhibit enzyme activity (14,21) and therefore probably also to inhibit the exchange reaction. NO could block N02/NO- 15N exchange without necessarily abolishing NO/H20-"0 ex- change if NO prevented access of nitrite to the active site. In addition, a small but unavoidable chemical NO;/NO-15N ex- change due to residual O2 contamination decreased the sen- sitivity of these experiments. O2 in presence of excess NO results in formation of N203 which partitions between nitrite exchange and irreversible hydration (2).

I t is of mechanistic interest to know whether nitrosation could inhibit reduction with which it presumably competes. The results are inconclusive on this point, because the nitro- sation rates were at most only a few per cent of the reduction rate.

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Supplementary Material to APPEN3IX C

ChTALYSIS OF NITROSYL TRANSFER REACTIONS BY A DISSIMILhTORY NITRITE REDWCTASE (CYTOCHROME c,al)

Choong-Syun X l m and Thomag C. Bollocher

APPENDIX A

lalculstlDn of the Amount of a Gas Species in the Face of Prior Sampling Lossea Cauaed by the Dlrect Expaneion llethod af Sampling

The ~ucceesive removal of e portion of the g85 phaae ever the reection mixture can be diagramed as follows

where P, and Ai are the mounts of the particular gas and of argon

Xi ie the mount of the particular gas removed ~n the i-th sample and Yi is respectluely. inferred to be in the v i a l (LB the remit of the I-th'sampling;

the amount of the particular gas produced in the interval betwen'the 1-th and the (irllth samples.

was 3951455 = 0.80 = 0.67lf0.67 + 0.17). The assumption that the nitrlte pool , m o r oration into a l l corn ounds (dissociation plus dehydietion and reduction)

assumDtian is tantemount to restrictlnn the modo1 to events at t i 0. The 18 infinitely large aseuree that 180 enters the p o o l irreversibly. This

model-is truncated at the third dehydration. because it ie clear that reduction or dissociation of an enzyme-bound nitrite molecule is much more likely than a fourth dehydration.

E' +

E' +

NO+ __f

0.58 E D +

E D +

E o +

N O , N20

N l 8 0 , N 2 ' 0 APPENDIX B

Esttmation of the Initial Rete of Ultrosation of Eydraeine by [15N]nitrite 88

Catalyzed by nitrite Reductase N1'O2- e E' 0.67

E' + 0.58 Under reddy acidic conditions, the nitroeation of hydrazine yield8

a z i d e . N20 and -onium (58, 59). Azide is Bubaequently converted to Up0 by nitroeation, but -onium is relatively stable. The reactions are depicted in E W . ~t involving ~ 1 5 ~ 0 ~ .

E D +

E o +

NO+ __f

N l 8 0 + 0.58

E' + N1'O, N;'O E O +

Scheme c1

would be the product of the indlvidval partition fractions along the path. The probability sum over all paths leading to 180-containing reduction products 18 0.050. and that over a l l paths leading to reduction Product8 irrespective of 0-isotope is 0.675. T e

ratio of theae two value* 0.074 is an e?@ate of Value of 0.014 derived Piam the model agrees reasonably well With2the data OP the atom fraction of lk0 in redaction product* at I = 0 i; loo$ H The

Fig. 4 from which an *torn fraction of 0.10 0.5 x 100/30) can be inferred for the reduction products et t = 3 Ln 100% H2lA0.

The probability of fallowing any aingle path io the partition network would be the product of the indlvidval partition fr.8Ctiom along the path. The probhbility sum over all paths leading to 180-containinp, reduction Droducts 18 0.050. and that over a l l 0-isotope is 0.675.

value of 0.014 derived Pi the atom fraction of % Fig. 4 from which an l80 for the reduction producl

The probability of fallowing any aingle path io the partition network

events. Thus, once nltrite is activated (dehydrated: it Stands s~milar The chief importance of the model 15 the relative rates of the various

chancee of being reduced or rehydrated. But once rehydration occurs the more probable event is dlssociation of 180-labeled nitrite rather than acLivat1on f o r B eecond tlme and reduction. Hydration can clearly compete with reduction. but the content of the reduction products is rather f a r f rom being in equilibrium with AZi80.

events. Thus, once nltrite is activated (dehydrated: it Stands s~milar The chief importance of the model 15 the relative rates of the various

chancee of being reduced or rehydrated. But once rehydration occurs the more probable event is dlssociation of 180-labeled nitrite rather than acLivat1on f o r B eecond tlme and reduction. Hydration can clearly compete with reduction. but the content of the reduction products is rather f a r f rom being in equilibrium with AZi80.

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C H Kim and T C Hollocher(cytochrome c,d1).

Catalysis of nitrosyl transfer reactions by a dissimilatory nitrite reductase

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