The catalytic mechanism of Pseudomonas cytochrome c peroxidase

ARCHIVES OF BIOCHEMISTRY Vol. 20’7, No. 1, March, pp.

The Catalytic

AND BIOPHYSICS 197-204, 1981

Mechanism

MARJAANA

of Pseudomonas

RoNNBERG,*,’ AND

Department

of Chemistry, Biochemistry,

University University

Cytochrome

TSUNEHISA ARAISO,‘,* H. BRIAN DUNFORD

of Alberta, of Helsinki,

Edmonton, Unioninkatu

Received

July

NILS

c Peroxidase ELLFOLK,”

Alberta T6G 2G.2, Canada, and *Department 35, SF-001 70 Helsinki 17, Finland

of

29, 1980

The catalytic mechanism of Pseudomonas cytochrome c peroxidase has been studied using rapid-scan spectrometry and stopped-flow measurements. The reaction of the totally ferric form of the enzyme with HzOz was slow and the complex formed was inactive in the peroxidatic cycle, whereas partially reduced enzyme formed highly reactive intermediates with hydrogen peroxide. Rapid-scan spectrometry revealed two different spectral forms, one assignable to Compound I and the other to Compound II as found in the reaction cycle of other peroxidases. The formation of Compound I was rapid approaching that of diffusion control. The stoichiometry of the peroxidation reaction, deduced from the formation of oxidized electron donor, indicates that both the reduction of Compound I to Compound II and the conversion of Compound II to resting (partially reduced) enzyme are one-electron steps. It is concluded that the reaction mechanism generally accepted for peroxidases is applicable also to Pseudomonas cytochrome c peroxidase, the intramolecular source of one electron in Compound I formation, however, being reduced heme c.

Cytochrome c peroxidase (ferrocytochrome c-%l:H,O, oxidoreductase, EC 1.11.1.5) from the denitrifying bacterium Pseudomonas aeruginosa is a hemoprotein containing two covalently bound heme c moieties in a single polypeptide chain (1,2). The enzymle catalyzes the peroxidation of Pseudomonas c-type cytochromes and azurin by hydrogen peroxide (3, 4). Unlike other peroxidases, e.g., those from yeast and horseradish, the reduced enzyme shows an absorption spectrum of the hemochrome type (1) which is characteristic of a lowspin hexacoordinated structure. However, circular dichroism (5) studies on the enzyme indicate that only one of the hemes is in a low-spin state while the other seems to be in a high-spin state. Titration of Pseudomonas cytochrome c peroxidase with CO and H,O, provides evidence that only the high-spin h’eme is accessible to ligands (6). The low-spin heme seems to form a cyto-

chrome unit which is capable of electron transfer (7) whereas the high-spin heme has the peroxidative function (6). It has been concluded from steady-state kinetic experiments that the enzyme is reduced before peroxidation of the electron donor begins (7). In this paper we characterize the catalytic mechanism of the enzyme. It will be shown that the totally ferric form of the enzyme, although forming a peroxide complex, is peroxidatically inactive whereas a partially reduced enzyme form appears to be enzymatically active, forming Compound I with hydrogen peroxide. Certain aspects of Compound I formation have been described in a preliminary communication (8). The primary compound is subsequently converted to Compound II. Despite the pronounced structural differences between Pseudomonas cytochrome c peroxidase and classical peroxidases, it is shown that the catalytic mechanism of the bacterial enzyme is closely similar to that of peroxidases in general.

1 Recipient of a Postdoctoral Fellowship from the Natural Sciences and Engineering Research Council of Canada. * Present address: Department of Biochemistry, University of Illinois, Urbana-Champaign, Ill. 61801.

MATERIALS Pseudomonas prepared from 197

AND

cytochrome acetone-dried

METHODS c peroxidase cells of P. aeruoinosa

was as

0003-9861/81/030197-08$02.00/0 Copyright All rights

0 1981 by Academic Press, of reproduction in any form

Inc. reserved.

198

RGNNBERG

described previously (1, 9). The preparation was homogeneous in disc electrophoresis and also in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The ratio A,,, nmlA080 nm of the preparation was 4.6. The concentration of a stock solution of the enzyme was determined spectrophotometrically using A (l%, 1 cm) equal to 12.1 at 280 nm (2). A molecular weight of 43,200 estimated from the iron content of the dry enzyme (2) was used to calculate the molar concentration of the peroxidase. Additional concentration determinations were made using E = 237 mM-' cm-’ for the oxidized enzyme at 40’7 nm (10). Cytochrome c-551 and azurin were prepared from acetone-dried cells of P. aeruginosa according to the procedures of Ambler (11) and Ambler and Brown (12), respectively. The purified cytochrome had an absorbance ratio A,,, nm (reduced)/A,,, nm = 1.2 and azurin had a ratio A,,, “,,, (oxidized)A,,, “,,, = 0.40. The concentration of cytochrome c-551 was obtained by using the value he (551 nm) (reduced-oxidized) = 19.0 mu* cm-’ (3). Azurin concentrations were determined by using ~(625 nm) = 5.1 mM-' cm-’ for the oxidized protein (3). Reduced electron donors were obtained by reduction with solid sodium dithionite and excess reductant was removed by dialysis. Reagent-grade potassium ferrocyanide from Fisher Scientific Company (Springfield, N. J.) was used without further purification. Hydrogen peroxide solutions were prepared from 30% hydrogen peroxide (Fisher Scientific Co.) and the concentration was determined spectrophotometrically at 230 nm using E = 72.4 M-’ cm-’ (13). The absorption spectra of the protein solutions were measured with a Cary 219 recording spectrophotometer. Stopped-flow and rapid-scan measurements were made on a Union Giken Model RA601 stopped-flow spectrophotometer equipped with a l-cm observation

ET AL. cell thermostated at 25 + 0.2”C. The rapid-scan absorption spectra are measured by means of a multichannel photodiode with a speed of 96 nm/ms and are memorized in a digital computer system. The analog replica is obtained afterward on an X-Y recorder. Spectra can be recorded during, immediately after, and at present time intervals after the stopped-flow mixing process. The nitrogen-gas-driving pressure used for these experiments yielded a dead time of 1.5 ms. RESULTS

Effect of Hydrogen Peroxide on the Totally Ferric Form of Pseudomonas Cytochrome c Peroxidase

Addition of hydrogen peroxide to a solution of ferric cytochrome c peroxidase resulted in increased absorbance at 407 nm, indicating the formation of a peroxide complex (Fig. 1). The time course of the reaction showed the complex formation to be first order with a rate constant of 7 s-l. The rate of the reaction was independent of H,O, concentration. The primary complex was slowly converted to a secondary complex in a reaction which was also independent of H,Oz concentration. However, there was a lag phase during which no reaction occurred for low concentrations of hydrogen peroxide. The half-life of this second reaction was about 16 s. The enzymatic activity of the peroxideferric enzyme complex was tested using rapid-scan spectrometry (Fig. 2). Ferric

Time (set)

FIG. 1. Curve for the formation of peroxide complex of ferric Pseudomonas cytochrome c peroxidase at 407 nm. Final concentrations of reactants: 2 pM enzyme and 3 pM hydrogen peroxide in 0.025 M sodium phosphate buffer, pH 6.0. Inset: Difference spectra of peroxide complex and ferric enzyme (a) 130 ms and (b) 300 s aRer mixing.

Pseudomonas CYTOCHROME

c PEROXIDASE

CATALYTIC

199

MECHANISM

enzyme 1 (PM) was mixed with an equimolar amount of H,O, and after 20 s reduced cytochrome c-551 (1 PM) was added. From the time course in Fig. 1 it was estimated that an incubation period of 20 s would give a mixture of “primary” and “secondary” complexes. Addition of reduced cytochrome did not affect the spectrum of the peroxide compound .within 130 ms. It may be concluded that the peroxide complex of ferric enzyme is peroxidatically inactive within this time range, and if a reaction occurs after this time limit it is so slow that its contribution to the peroxidative cycle is negligible. Effect of Hydrogen Peroxide on Pseudomonas Cytochrome c Peroxidase Partially Reduced with Reduced Axurin or Ferroc yanide

Contrary to the case of the totally ferric enzyme, partially reduced peroxidase appeared to react rapidly with hydrogen I

I

1

I

I

I

0.3

$

6 0.2 A 5 _i; Q 0.1

I

400 Wavelength

440 (nm)

FIG. 2. Rapid-scan spectra on the mixture of the peroxide complex of ferric Pseudomonas cytochrome c peroxidase and reduced cytochrome c-551. Enzyme and peroxide were incubated 20 s before mixing with reduced cytochrome c-551. (a) spectra of the mixture 2 and 130 ms after mixing; (b) spectrum of ferric enzyme; (c) spectrum of reaction mixture 2 ms after mixing minus spectrum of ferrous cytochrome c-551; (d) spectrum ofreduced cytochromec-551; (e) spectrum of oxidized cytochrome c-551. All concentrations 1 FM in 0.025 M sodium phosphate buffer, pH 6.0.

0’

’

I

I

400 Wavelength

I

440 (nm)

FIG. 3. Rapid-scan spectra on the formation of Compound I and Compound II from partially reduced Pseudomonas cytochrome c peroxidase. A: (a) spectrum of partially reduced enzyme; (b, c) spectrum showing Compound I formation 1 and 3 ms, respectively, after mixing with H,O*. Final concentrations: 1 yM enzyme, 4 pM azurin, 2 pM hydrogen peroxide in 0.025 M sodium phosphate buffer, pH 6.0. B: (a) spectrum of partially reduced enzyme; (d) spectrum showing Compound I formation 1 ms after mixing; (e) spectrum showing Compound II formation obtained 30 ms after mixing; (f) spectrum of the enzyme which returned to the partially reduced form 760 ms after mixing with hydrogen peroxide. The spectra are shifted to lower absorbances for clarity. Final concentrations: 1 pM enzyme, 10 pM azurin, and 2 pM hydrogen peroxide.

peroxide to form an active intermediate in the peroxidatic cycle. Rapid-scan spectra on the mixture of partially reduced Pseudomonas cytochrome c peroxidase and hydrogen peroxide are given in Fig. 3. In the presence of a fourfold excess of reduced azurin over enzyme the addition of H,O, results in formation of an intermediate within a few milliseconds. The intermediate has an absorption maximum at 411 nm and isobestic points with the partially reduced enzyme at 401, 415, and 429 nm (Fig. 3A). Three milliseconds after mixing the absorp-

RGNNBERG

200

ET AL.

tive form of the enzyme from which Compound I is derived as partially reduced. Kinetics of Formation of Compound I.

’ [Az(I)]initi.l

/ [‘,nz,me]

3

FIG. 4. Formation of Compound I from Pseudomonos cytochrome c peroxidase which has been partially reduced with varying amounts of reduced azurin (AZ(I)). Enzyme concentration, 1 pM; hydrogen peroxide concentrations, 0.8, 0.8, 1.0, 1.2, and 1.2 j&M, in the order of increasing azurin concentration, in 0.025 M sodium phosphate buffer, pH 6.0.

tion of the intermediate is increased but the isosbestic points are unchanged, suggesting the presence of one intermediate only. Using a higher electron donor concentration two different spectral forms are observed (Fig. 3B). Within 1 ms after mixing the spectrum of the primary intermediate is observed but within 30 ms the spectrum has changed to a form which is isobestic with partially reduced enzyme at 414 nm. The spectrum of partially reduced enzyme is regained 760 ms after mixing. We refer to the half-reduced enzyme form as resting enzyme. The formation of the primary compound between hydrogen peroxide and the partially reduced enzyme was measured in titration experiments at 413 nm, the isosbestic point of the totally reduced and totally oxidized enzyme. When the formation of the compound was maximal, the extrapolated value for the ratio of reduced azurin to enzyme was found to be 1:l (Fig, 4), which gives about half-reduced enzyme. Higher reduction of the enzyme resulted in a decrease in the formation of the primary compound. It appears that in the halfreduced enzyme one of the heme groups is essentially reduced while the other is mainly in the ferric state (unpublished results). Since in the experiments reported here the exact amount of Compound I formation is unknown we refer to the reac-

and Decomposition

The kinetics of Compound I formation was followed at 413 nm and a very rapid reaction was observed. For example, if a lofold molar ratio of azurin to totally oxidized enzyme (allowed to sit for approximately 20 min to obtain the partially reduced form) is mixed with a 2-fold molar ratio of H,O, to enzyme, maximal Compound I formation is observed within 5 ms. This rapid increase was followed by a slower reaction which corresponded to the decomposition of Compound I. A second-order rate constant, 1.2 X lo8 M-’ S-‘, for the formation of Compound I was calculated from a plot of observed pseudo-first-order reaction rate, V ohs, divided by the concentration of partially reduced enzyme versus H,O, concentration (Fig. 5) using the equation V ohs = k 1[H,O,] [partially reduced enzyme]. Decomposition of Compound I by azurin was followed in stopped-flow experiments where 2.05 f..&Menzyme was incubated with different concentrations of reduced azurin

3 003

~w*linitial (PM)

FIG. 5. A plot for the determination of the secondorder rate constant k, for the rate of Compound I formation. Observed reaction rate (A absorbance at 413 nm) divided by the concentration of partially reduced enzyme is plotted against the concentration of hydrogen peroxide. Reaction conditions: 0, 0.5 FM enzyme, 30 PM ferrocyanide; 0, 1 PM enzyme, 0.4-1.6 PM azurin, all in 0.025 M sodium phosphate buffer, pH 6.0. The points represent the mean values of five measurements, the error being ? 10%.

Pseudomonas

CYTOCHROME

c PEROXIDASE

(lo-60 PM) and 10 j..kM H,O, was added. The observed k values (Fig. 6) are linearly dependent on azurin concentration, and the plot gives a second-order rate constant of about 2 x 10’ M-’ s-l. Decomposition of Compound I may be correlated with the formation of Compound II. Kinetics of the Reaction of Compound II with Axurin

Kinetics of the decomposition of Compound II was followed in the steady state by measuring the formation of oxidized azurin at 580 nm where reduced and oxidized enzyme are isosbestic. The measurements were made on the stopped-flow apparatus using the i:nitial portion only of the reaction trace. From the reaction trace, the rate of product formation was 66.7 x lo+ ms-’ when [enzyme] was 1 PM, [H,O,] was 3 PM, and [reduced azurin] was 8 PM. The formation of oxidized azurin is described by v = d[ox azurinlldt

= k;[I]

= Z{(k;k;k;)l(k;k;

where

[I]

and

[II],

+ kA[II] + k;kj + kjk;)},

respectively,

are

1000

-

500

-

> [Azurin]

(PM)

FIG. 6. Plot of kabs, the rate constants for Compound I decomposition, versus azurin concentration measured at 415 nm which is the isosbestic point between Compound I and partially reduced enzyme. Pseudomonas cytochrome c peroxidase, 2.05 pM; hydrogen peroxide, 10 pM, in 0.025 M sodium phosphate buffer, pH 6.0.

concentrations of Compound I and Compound II of Pseudomonas cytochrome c peroxidase.

kl = k,PLXM

ks II + red azurin + E + ox azurin

Stopped-flow experiments were carried out with 1 FM enzyme, 10 FM reduced azurin, anld 2 PM hydrogen peroxide (final concentrations). At 413 nm a rapid increase in absorbance was observed cor-

201

7 25 B 4

I + red azurin 2 II

Stoichiometry of Compound I and Compound II Formation

MECHANISM

‘5oo I

k, E + H,O, + I

With k; = 1.2 x lo8 x 3 x lop6 and kl = 2 x lo7 x 8 x lO-‘j, k, was calculated to be equal to 6 x lo6 M-’ s-l, the value being an estimate, which should be accurate within a factor of 2, since the error in k, is a function of the cumulative errors in V, kl, and k,.

CATALYTIC

kl = k,[red

azurin]

kj: = k,[red

azurin]

responding to the formation of Compound I (Fig. ‘7A). At 415 nm (isosbestic point between Compound I and half-reduced enzyme) there was fist a rapid decrease which leveled off during the 50 ms when Compound I was decomposed. After that a steady increase corresponding to the formation of Compound II was observed. The secondary compound was formed much more slowly than Compound I; the maximal amount of Compound II being formed 300 ms after mixing. Formation of oxidized azurin was followed at 580 nm. When Compound I had decomposed, 1 pmol of oxidized azurin had

RGNNBERG

ET AL.

FIG. 7. Stopped-flow curves for the reaction between Pseudomonas cytochrome c peroxidase, reduced azurin, and hydrogen peroxide followed at different wavelengths. Reaction conditions: 1 pM enzyme and 10 pM azurin, incubated 20 min before mixing with 2 pM H,Oz (all concentrations final), in 0.025.~ sodium phosphate buffer, pH 6.0 (A) Reaction curve for the formation of Compound I followed at 413 nm, isosbestic point between reduced and oxidized enzyme. (B) Reaction followed at 415 nm, the isosbestic point between Compound I and partially reduced enzyme, corresponding to Compound II formation. (C) Formation of oxidized azurin followed at 580 nm, where oxidized and reduced enzyme are isosbestic. Arrows (a) and (b) show the formation of 1 and 4 pmol of oxidized azurin, respectively.

been formed (arrow (a) in Fig. W), indicating that the conversion of Compound I to Compound II is a one-electron step. By the end of the reaction 4 pmol of oxidized azurin has been formed (arrow (b) in Fig. 33). This is in accordance with two cycles of two successive one-electron steps taking place (i.e., conversion of Compound I followed by reduction of Compound II to resting enzyme), as the reaction mixture was 2 PM in H,O, and 1 PM in enzyme. DISCUSSION

The present results confirm earlier observations of formation of a complex between the totally oxidized enzyme and hydrogen peroxide (6, 10). The formation of the complex is rather slow, the half-life of the reaction being about 100 ms. The conversion of the primary complex into a secondary complex has a half-life of 16 s. The present results provide the first direct evidence that both of these species are catalytically inactive toward reduced substrates like cytochrome c-551. This is in agreement with earlier observations that the enzyme has

to be partially reduced before the peroxidatic oxidation of reduced substrates can be initiated (7). In contrast, when partially reduced enzyme reacts with hydrogen peroxide a highly reactive intermediate is formed. The rapidscan spectra show that the primary compound between the peroxidase and hydrogen peroxide is formed a few milliseconds after mixing. The second-order rate constant for the formation of the primary compound was calculated to be 1.2 x lo8 M-l s-l. The value is somewhat higher than that of other peroxidases. The literature values of the rate constant for the reaction between hydrogen peroxide and different peroxidases vary from 0.2 x lo7 (horseradish peroxidase, isoenzyme A2) (14) to 4.5 x lo7 M-l s-l (yeast cytochrome c peroxidase) (15). A second-order rate constant of this order of magnitude indicates that the combination of the reactants is nearly diffusion-controlled. The conversion of Compound I to Compound II is linearly dependent on the azurin concentration with a second-order rate constant of 2 x 10’ M-’ s-l. This value is somewhat higher than those found for yeast

Pseudomonas

CYTOCHROME

c PEROXIDASE

SCHEMEI. high spin,

The enzymatic cycle for horseradish R. to a free radical.

cytochrome c peroxidase when dicyano-bis(l,lO-phenanthroline) iron(I1) (16) and ferrocyanide (17) were used as reductants. For the conversion of Compound I + Compound II, one reducing equivalent was needed and a second one for the reduction Compound II + “resting” enzyme. Using the rate constants obtained above, it can be calculated that Compound I comprises 23% and Compound II 77% of the total amount of enzyme when the concentration of H,O, is saturating. The present results show that the reaction mechanism characterized as of modified ping-pong form, generally accepted for peroxidases, is applicable also to Pseudomonas cytochrome c peroxidase. From steady-state kinetic experiments, however, it was earlier suggested on the basis of intersecting reciprocal plots that the enzyme follows a sequential mechanism (4). Considering the correlation between maximal Compound I formation and concentration of reduced electron donor observed with transient kinetics, it is clear that intersecting plots in st.eady-state kinetics results from varying amounts of totally ferric, totally ferrous, a,nd partially reduced forms of the enzyme present in the steady-state mixture. In the enzymatic reaction of hydrogen peroxide with peroxidases like horseradish peroxidase two intermediate compounds can be isolated, the green primary Compound I and the red secondary Compound II. The 2-equivalent oxidation of the native enzyme to Compound I does not yield Fe5+ but Fe4+, with the extra electron abstracted from the protein or the porphyrin, resulting in the formation of a radical cation (R .) (18-22). The chromophores of the two compounds are electronically similar (23-27). The enzymatic cycle for horseradish perox-

peroxidase

CATALYTIC

(HRP);

203

MECHANISM

1s and hs refer

to low spin and

idase and most other peroxidases is as shown in Scheme I. The electrons for the reductive steps of the cycle are abstracted from the oxidizable substrates. A similar type of scheme can be given for the reaction of Pseudomonas cytochrome c peroxidase on the basis of the present results. It is assumed that in the half-reduced enzyme mainly the low-spin heme c is in the ferrous state and the highspin heme c in the ferric state. In horseradish peroxidase the addition of a neutral oxygen atom to Fe3+ would convert it to a formal Fe5+ species but an extra electron is supplied from elsewhere in the heme protein, the end product being Fe4+. In Pseudomonas cytochrome c peroxidase this electron is (Inactive CmpkxA

H,O, t

-complexes)

,,* =16r

Complex El

Cl1 Compound

I

c:s+ R

tA+ Is

CompoudE

FIG. 8. Reaction scheme for the enzymatic cycle of Pseudomonas cytochrome e peroxidase. R, reduced electron donor; hs, high spin; Is, low spin; the symbol c refers to a heme c group; c3+ in the resting enzyme refers to a conformational change or other difference from the same heme group in the totally ferric enzyme form. Rate constants, all in units of Mm1s-l: k, = 1.2 x IO*, k2 = 2 x lo’, and k, = 6 x lo6 for hydrogen peroxide and reduced azurin as oxidizing and reducing substrates.

204

RGNNBERG

supplied by the reduced heme c, the structure of Compound I being Fe4+-Fe3+. Scheme II, shown in Fig. 8, gives the enzymatic cycle of Pseudomonas cytochrome c peroxidase. Reduction of the totally ferric enzyme to the resting (half-reduced) form must cause a conformational change which makes the remaining ferric heme c group accessible to ligands and to hydrogen peroxide. This active form of the heme c group is designated as *C in Fig. 8. Finally by analogy with most other peroxidases we have designated Compound II in Fig. 8 as containing Fe4+. However, we have no proof that Compound II of Pseudomonas cytochrome c peroxidase is not *Ciz-Cyz. The equilibrium between two forms of Compound II in yeast cytochrome c peroxidase may be relevant (29). ACKNOWLEDGMENT A traveling Fennica to N.E.

grant from is gratefully

Societas Scientiarum acknowledged.

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ET AL. 9. SOININEN, R. (1972) Acta Chem. Scund. 26, 2535-2537. 10. SOININEN, R., AND ELLF~LK, N. (1973) Acta Chem. &and. 27,35-46. 11. AMBLER, R. P. (1963) Biochem. J. 89, 341-349. 12. AMBLER, R. P., AND BROWN, L. H. (1967) Biothem. J. 104, 784-825. 13. GEORGE, P. (1953) Biochem. J. 54, 267-276. 14. MARKLUND, S., OHLSSON, P.-I., OPARA, A., AND PAUL, K.-G. (1974) Biochim. Biophys. Acta 350, 304-313. 15. Loo, S. J., AND ERMAN, J. E. (1975) Biochemistry 14,3467-3470. 16. JORDI, H. C., AND ERMAN, J. E. (1974) Biochemistry 13, 3741-3745. 17. ERMAN, J. E. (1975) Biochim. Biophys. Actu 397, 36-42. 18. GEORGE, P. (1953) J. Biol. Chem. 201,427-434. 19. BRILL, A. S., AND WILLIAMS, R. J. P. (1961) Biochem. J. 78, 253-262. 20. SCHONBAUM, G. R., AND Lo, S. (1972) J. Biol. Chm. 247, 3353-3360. 21. PEISACH, J., BLUMBERG, W. E., WI~ENBERG, B. A., AND WI~ENBERG, J. B. (1968) J. Biol. C&m. 243, 1871-1880. 22. DOLPHIN, D., FORMAN, A., BORG, D. C., FAJER, J., AND FELTON, R. H. (1971) Proc. Nut. Acad. Sci. USA 68, 614-618. 23. MAEDA, Y., AND MORITA, Y. (1967) Biochem. Biophys. Res. Commun. 29680-685. 24. STILLMAN, M. J., HOLLEBONE, B. R., AND STILLMAN, J. S., (1976) Biochem. Biophys. Res. Commun. 72, 554-559. 25. Moss, T. H., EHRENBERG, A., AND BEARDEN, A. J. (1969) Biochemistry 8, 4159-4162. 26. SCHULZ, C. E., CHIANG, R., AND DEBRUNNER, P. G. (1979) J. Phys. C 40, 534-536. 27. SCHULZ, C. E., DEVANEY, P. W., WINKLER, H., DEBRUNNER, P. G., DOAN, N., CHIANG, R., RUTTER, R., AND HAGER, L. P. (1979) FEBS Lett. 103, 102-105. 28. DUNFORD, H. B., AND STILLMAN, J. S. (1976) Coord. Chem. Rev. 19, 187-251. 29. COULSON, A. F. W., ERMAN, J. E., AND YONETANI, T. (1971) J. Biol. Chem. 246, 917-924.