Horseradish peroxidase-catalyzed aerobic oxidation and peroxidation of indole-3-acetic acid

ARCHIVES

OF BIOCHEMISTRY

AND BIOPHYSICS

Vol. 296, No. 1, July, pp. 27-33, 1992

Horseradish Peroxidase-Catalyzed Aerobic Oxidation and Peroxidation of Indole-3-Acetic Acid I. Optical Spectra Diana Metodiewa,* Giuseppe Cilento,

Mariza Pires de Melo, Jorge A. Escobar, and H. Brian Dunford*,1,2

*Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2; and Institute University of Sco Paulo, C.P. 20780, 01498 S&o Pa&o, Brazil

Received December 2, 1991, and in revised form February

23, 1992

A study of the indole-3-acetate reaction with horseradish peroxidase, in the absence or presence of hydrogen peroxide, has been performed, employing rapid scan and conventional spectrophotometry. We present here the first clear spectral evidence, obtained on the millisecond time scale, indicating that at pH 5.0 and for high [enzyme/ substrate] ratios peroxidase compound III is formed. Most, if not all, of the compound III is formed by oxygenation of the ferrous peroxidase. There is an inhibitory effect of superoxide dismutase and histidine on compound III formation which indicates the involvement of the active oxygen species superoxide and singlet oxygen. It is concluded that the oxidation of indole-3-acetate by horseradish peroxidase at pH 5.0 proceeds through compound III formation to the catalytically inactive forms P-670 and P-630. A reaction path in which the enzyme is directly reduced by indole-3-acetate might be involved as an initiation step. Rapid scan spectral data, which indicate differences in the formation and decay of enzyme intermediate compounds at pH 7.0, in comparison with those observed at pH 5.0, are also presented. At pH 7.0 compound II is a key intermediate in oxidation-peroxidation of substrate. Mechanisms of reactions consistent with the experimental data are proposed and discussed. 0 1992 Academic Press, Inc.

The physiologically oxidation/peroxidation

important peroxidase-catalyzed of the plant hormone IAA3 is a

i On study leave at the University of ,550 Paulo. ’ To whom correspondence should be addressed at the Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2. 3 Abbreviations used: IAA, indole+acetic acid; HRP, horseradish peroxidase; HRP-I, HRP-II, and HRP-III, compounds I, II, and III of 0003.9861/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

of Chemistry,

very complex process as inferred from the multitude of products formed and the number of enzyme conversions (HRP-I, II, and III; inactive forms P-670, P-678, P-630). The observed behavior is highly dependent upon experimental conditions (l-lo). To date there have been two principal methods of investigation: (a) analysis of the stable end products (6) and/or free radical intermediates (8,9); (b) kinetic investigations at room (2-5) or low temperature (lo), performed on a second or minute time scale. Despite extensive studies since 1955 (l), the HRP/IAA reaction is far from being completely understood: some proposals are still speculative and several mechanistic questions remain unanswered (11). It has been proposed (2) that a shift between oxidation and peroxidation reaction cycles exists and that it is induced by variations in factors such as the HRP/IAA ratio and pH (4). According to these suggestions, a high ratio promotes essentially the peroxidase cycle; a low ratio (at acidic pH values) leads to the involvement of HRP-III (oxyperoxidase) (4). An early suggestion was that the initial step in the HRP-catalyzed oxidation of IAA is a one-electron transfer process resulting in Fe”-HRP formation and oxygen binding to the reduced enzyme (oxygenase-like reaction) (2,4). This process is of great importance for the regulation of plant growth at the molecular level (2). HRP reduction by solvated electrons (11) and oxygen binding to Fe”-HRP are very fast processes (12). With the exception of one series of four spectral traces [Fig. 7 of Ref. (3)] we present for the first time optical spectral studies on the millisecond time scale. Two different pH values

HRP; Fe”-HRP, ferrous HRP, R.Z., reinheitzahl, purity number, ratio of absorbances at 403/280 nm; P-670 (ferric) compound (verdohemoprotein) formed from HRP with absorbance peak at 670 nm, also called compound IV, P-678 (ferrous) compound of HRP; P-630, another inactive compound formed from HRP; SOD, superoxide dismutase. 27

28

METODIEWA

and high enzyme/substrate ratios were employed. Because the use of rapid scan spectral measurements, a precise detection method for enzyme conversions, requires a high concentration of HRP, only measurements at relatively high enzyme/substrate ratios could be conveniently carried out. We compare enzyme conversions in the oxidation of IAA by native enzyme or by its peroxidation by preformed HRP-III. Our results provide some new insights into the relations between these two cycles (8). We also present the first optical spectral evidence, obtained in millisecond reaction times, to indicate that the peroxidase cycle does not function during IAA oxidation at low pH and high enzyme/substrate ratio. Rather, direct oxygen activation is involved. Using the specific scavengers SOD and histidine we provide evidence for the involvement of 0; and ‘OZ. The system emits light even when the enzyme concentration is very low. The light emission and oxygen uptake studies are reported in the following paper. MATERIALS

AND METHODS

HRP, Grade 1, was purchased from Boehringer-Mannheim Corp. as an ammonium sulfate suspension and extensively dialyzed against deionized water. The R.Z. of the resultant solution was 3.4. The concentration of HRP was determined spectrophotometrically at 403 nm using a molar absorptivity of 1.02 X 10’ M-l cm-’ (13). SOD was obtained from Sigma and its concentration was calculated using a molar absorptivity of 15.9 mM-’ cm-’ at 265 nm (14). HRP-III, stable for 12 min, was prepared using a 250 molar excess of hydrogen peroxide in citrate buffer (pH 5.0, 50 mM). IAA (Sigma) was purified by sublimation in high vacuum at 130-135°C. The white crystalline powder has a m.p. of 16%169’C. No difference in light emission intensity and/or kinetics was observed after purification. A stock solution of IAA (10 mM, also in citrate buffer) was prepared. HRP-I, stable at least 6 min, was prepared using a 1 molar equivalent of hydrogen peroxide in the same citrate buffer. IAA was added to a final concentration of either 0.5 mM or 50 pM for reaction with HRP-III. For time measurements greater than 10 s the spectra were scanned from 700 to 350 nm using a Cary 219 spectrophotometer. For shorter times spectral measurements were made on a Photal (formerly Union Giken) Model 601 Rapid Reaction Analyzer over a 96nm range. One reservoir contained HRP-III and the other IAA. Similar rapid scan experiments were performed using either native HRP or HRP-I in one reservoir and either IAA or IAA + HzOz in the other. Experiments at pH 7.0 were conducted in a manner identical to those at pH 5.0, except that 50 IIIM potassium phosphate buffer was used.

ET AL.

Pz 0.15 2 8 4 0.10 2 ‘G s 0.05 d

0Do~

j

380

400

4m 440360 380 Wavelength (nm)

400

420

440

I

FIG. 1. Rapid-scan spectra in the Soret region of an intermediate of HRP (3.2 PM) accumulated during oxidation of IAA (5.0 mM) at pH 5.0 (50 mM citrate buffer). A, in the absence of SOD. B, in the presence of SOD (100 nM). Scans a-f-115, 170, 280, 390, 500, and 830 ms after mixing.

the 533-nm isosbestic point observed during formation of HRP-III. The isosbestic point between HRP and HRPII at 408 nm in the Soret (Fig. 1A) is shifted to 410 nm. Therefore, in this system at least three species and two reactions occur in the conversion of native HRP to HRPIII by IAA. As can be seen in Figs. 1B and 2B, the presence of SOD (100 nM) in the reaction mixture causes an inhibition of HRP-III formation. In the presence of SOD the isosbestic point at 408 nm shifted to 412 nm (Fig. 1B). In the visible region formation of the HRP-III precursor occurs rapidly during the first 60 ms and is complete after 280 ms (Fig. 2B). It can be seen from Fig. 3 that histidine slows the reaction. The spectrum of the

1

RESULTS Oxidation

of IAA by HRP or Preformed HRP-III

Oxidation of IAA by HRP atpH 5.0. As shown in Figs. 1 and 2, between 60 and 630 ms after mixing of IAA with HRP a new spectral species appears with absorption

maxima at 418 nm (Soret region, Fig. 1) and at 543 and 577 nm (visible part, Fig. 2). Isosbestic points occur at 410 and 533 nm. The spectrum after 830 ms (Figs. 1A and 2A) has all the characteristics of HRP-III (11). The native enzyme spectrum (Fig. 2A) does not coincide with

540 560 560 Wavelength (nm)

61

FIG. 2. Rapid-scan spectra in the visible region of an intermediate of HRP (10.8 FM) accumulated during oxidation of IAA (5.0 mM) at pH 5.0 (50 mM citrate buffer) A, in the absence of SOD. B, in the presence of SOD. Scan a, no IAA, Scans b, 60; c, 390, d, 500; and e, 830 ms after mixing. Scans f, g, 1 and 2 min after mixing.

OPTICAL

o.ooi

SPECTRA

OF PEROXIDASE/INDOLEACETIC

I 360

403

420

440

380

420

400

I I ow3M

440

FIG. 3. Effect of singlet oxygen quencher histidine (6.0 mM) on the rapid-scan Soret spectra of the reaction of HRP (1.2 pM) with IAA (2.5 mM) at pH 5.0. A, control (no histidine); scans a-d, 100, 200, 400, and 800 ms after mixing. B, conditions same as in A, except 6.0 mM histidine added with IAA; scans a-b, 100 and 800 ms after mixing.

native enzyme clearly passes through the 410-nm isosbestic point. The spectral changes of HRP observed from 20 s until 15 min after mixing with IAA are shown in Fig. 4. Between 20 s and 12 min after mixing of HRP with a lo-fold excess of IAA, one can observe only P-670 formation (Fig. 4A). Surprisingly, when HRP is mixed with a loo-fold excess of IAA, the simultaneous formation of P-630 and P-670 is evident from 20 s until 15 min (Fig. 4B). Oxidation of IAA by preformed HRP-III at pH 5.0. In Fig. 5 rapid scan results are displayed on the decay of HRP-III in the presence of IAA. From 0.6 s up to 8.3 s after mixing, the HRP-III Soret peak decays with isosbestic points at 381 and 439 nm (Fig. 5A). The same decay rate was observed in the presence of 50 ELM or 0.5 mM IAA. The family of visible spectra do not reveal a well-defined isosbestic point (Fig. 5B). It is tempting to speculate that the species being formed from HRP-III (Figs. 5A and 5B) is a precursor of P-670. There is support for this postulate in the spectral results obtained on longer time scales where the P-670 spectrum is obvious (Fig. 6). It is evident that the extent and rate of P-670 formation is dependent upon IAA concentration. Intermediate for-

0.60

420

I Oca

MO

I 520

540

580

562

Wavelength

(nm)

6x

(nm)

FIG. 5. Rapid-scan spectra of HRP-III decay in presence of IAA (0.5 mM) (pH 5.0, 50 mM citrate buffer). A, Soret; B, visible part. Scans ac-0.6, 6.1, and 8.3 s after mixing. The same rate of decay of HRP-III was observed in the presence of 50 pM IAA.

mation of HRP-II is not observed until 15 min after mixing of HRP-III with IAA (Fig. 6). The increase of absorbance at 550 nm, simultaneously with P-670 formation, indicates the presence of a stable ferrous form (Fig. 6B). In the absence of IAA, the spontaneous decay of HRPIII (Fig. 7) clearly indicates the formation of HRP-II, stable for 60 min, and P-670. Oxidation of IAA by native HRP at pH 7.0. The surprising results of the IAA reaction with HRP at pH 7.0 are depicted in Fig. 8. Within 0.6 s after mixing, a new spectral species starts to form with absorption maxima at 420 nm, and the reaction is complete in about 8.3 s (Fig. 8A). An isosbestic point at 410 nm is apparent (Fig. 8A). To characterize the new species further, its formation was followed in the visible region (Fig. 8A). The newly formed intermediate does not reveal the peaks ascribed either to HRP-II or to HRP-III. The broad shoulder at 557 nm (Fig. 8A) indicate traces of Fe”-HRP present as an intermediate species. In the presence of SOD the family of spectra is not isosbestic with the native enzyme, the absorptivity at 420 nm increases while the isosbestic point at 410 nm is maintained (Fig. 8B). The increase of absorption at 527 and 555 nm indicates the formation of some HRP-II.

080

008

060

006

040

004

0.06 0 e

0.40

0.04

2 B 2

403 Wavelength

29

REACTION

I ml

Wavelength (nm)

d c

ACID

2 2b

a 0.20

0.02

400

450

500

Wavelength

550

(nm)

600

650

0.W 700

0.00L’ 350

I 400

450

500

550

800

650

700

Wavelength (nm)

FIG. 4. Spectral changes observed on a longer time scale in the reaction of HRP (4.7 PM) with IAA (pH 5.0, 50 mM citrate buffer). Scan a, no IAA present. A, 50 pM IAA, scan b, 20 s, scans c and d, 6 and 12 min after mixing. B, 0.5 mM IAA; scan b, 20 s, c-f-3, 6, 12, and 15 min after mixing.

30

METODIEWA

I.00

;

loo

0.10

0.60

0.06

0.10

A

0.60

0.06

0.60

0.06

2 8

ET AL.

i:;

a

0.00 350

i

b d.0

400

0.06

2 0.40

0.04

0.02

0.20

0.02

0.W 7w

0.00

0.04

2 0.40 0.20

4gi 0.60

450

5w Wavelength

554

600

650

(nm)

350

400

450 500 Wavelength

550

wo

650

0.00 700

(nm)

FIG. 6. Absorption spectra of an intermediate accumulated during IAA oxidation by HRP-III at pH 5.0. A, 0.5 mM IAA, scan a, 5.3 pM native HRP as reference, b, HRP-III 10 min after formation; c, 20 s; d and e, 3 and 6 min after IAA addition. B, 50 pM IAA; scan a, 4.7 @M native HRP as reference; b, HRP-III 10 min after formation, scans c-e-20 s, 150 s, and 15 min after IAA addition.

When HRP was mixed with approximately a lo-fold (data not shown). SOD slightly enhances HRP-II forexcess of IAA, the formation of HRP-II (stable for 10 mation. min) and P-670 (stable for 60 min) is obtained (Fig. 9A). With a loo-fold excess of MA formation of HRP-II as a DISCUSSION transient species observable 20 s after mixing and stable Oxidation of IAA by HRP at pH 5.0 formation of P-670 occurs (Fig. 9B). The presence of 100 The literature on the IAA/HRP reaction is not in nM SOD in the reaction mixture causes stabilization of HRP-II, still observed 6 min after the start of the reaction, agreement, particularly over the role of Fe”-HRP and HRP-III. Ricard and co-workers were the first to postulate and enhanced P-670 formation (data not shown). Oxidation of IAA by preformed HRP-III atpH 7.0. The an important role for HRP-III (2). Yamazaki’s group has autodecay of HRP-III to HRP-II and P-670 at pH 7.0 in maintained that at a high enzyme/substrate ratio Fe”the absence of IAA is similar to results at pH 5.0 (data HRP and HRP-III are not involved, and that the oxinot shown). In the presence of IAA disappearance of dation of IAA proceeds by a normal peroxidase cycle inHRP-III is faster at pH 7.0 than at pH 5.0 as was observed volving HRP-I and HRP-II (3,15). Smith et al. proposed in a low temperature investigation (10). It leads to HRP- that a Fe”-HRP F? HRP-III shuttle is operating at low enzyme/substrate ratio and the normal peroxidatic cycle II and P-670 formation; 100 nM SOD or 10 mM histidine proceeds at high enzyme/substrate ratio (4). had no effect (data not shown). Our spectral results in Figs. 1-4, registered for high enzyme/substrate ratio, largely obtained on a faster time Peroxidation of IAA by HRP and H202 scale than nearly all previous work, lead to some new conclusions. We show that the normal peroxidase cycle Peroxidation of IAA by HRP and HzOz or by preformed HRP-I at pH 5.0. Formation of HRP-I from H202 and does not function at low pH and high enzyme/substrate native HRP is shown in Fig. 10. The reaction is complete in 80 ms. The reaction of native HRP with both Hz02 and IAA, also over a 80-ms time scale, is shown in Fig. 10B. The results in Fig. 10B indicate a biphasic process: scans a and b intersect at 410 nm whereas scans c and d intersect at 408 nm. The conversion HRP --* HRP-I --* HRP-II followed by HRP-III formation is being observed. SOD does not affect these transformations. Both HRPII and HRP-III formation are observed in the direct reaction of preformed HRP-I with IAA with no isosbestic points observed (Fig. 11). Peroxidation

of IAA

by HRP

and

Hz02

at pH

7.0. Visible region rapid scan spectra obtained by mixing 3.9 PM native HRP with both 15.0 PM H202 and 2.5 mM IAA showed HRP-II formation over a period of 830 ms

FIG. ‘7. Absorption spectra of an intermediate accumulated during autodecay of HRP-III (in the absence of IAA) at pH 5.0. Scan a, 4.6 pM native HRP as reference; b, HRP-III 10 min after formation; c, 60 min after formation.

OPTICAL

000 360

360

400

420

440

SPECTRA

520

540

OF PEROXIDASE/INDOLEACETIC

560

580

6W

0.00

0.00

360

360

ACID

REACTION

403

420

Wavelength (nm)

31

440 520 Wavelength (nm)

540

560

560

6W

0.00

FIG. 8. Rapid-scan spectra of an intermediate of HRP accumulated during oxidation of IAA at pH 7.0 (A) and effect of SOD (100 nM) (B). HRP, 3.5 pM (Soret, A and B, left) and 7.1 pM (visible part A and B, right); IAA 2.5 and 5 mM, respectively (50 mM potassium phosphate buffer). Scan a, native HRP; b-d-0.6, 2.8, and 8.3 s after mixing.

ratio. The direct activation of molecular oxygen is an important feature of the reaction of IAA with HRP. HRPIII, which is converted to P-670 is formed; it is not converted back to Fe”-HRP. Data presented in Figs. 1A and 2A provide the first clear spectral evidence indicating the two-step formation of HRP-III in the spontaneous oxidation of IAA by HRP. Formation of HRP-III as a product is clearly observed, but the isosbestic point between native HRP and HRPIII is shifted, indicating formation of an intermediate species. The results can be accounted for by the following: HRP + IAA % Fe”-HRP + IAA’

PI

Fe”-HRP + O2 + HRP-III

PI

The equilibrium in Eq. [l] must exist, however unfavorably. This is in accordance with the laws of thermodynamics. The question then becomes the following: is the concentration of Fe”-HRP formed in Eq. [l] sufficiently large to initiate the reaction? It is instructive to explore this possibility. Removal of Fe”-HRP in Eq. [2] would help to drive the equilibrium in Eq. [l] to the right. Observation of the direct reduction of HRP by IAA, Eq. [l], was claimed by Ricard and Nari (16) under anaerobic conditions. This result was confirmed by Smith et al. (4). Nakajima and Yamazaki (3) did not observe direct reduction of native HRP by IAA, under conditions

made anaerobic by addition of an oxidase with its substrate plus catalase, but did observe reduction in later stages of the reaction which they attributed to the reaction HRP + IAK + Fe”-HRP + IAA+

[31

Thus there now appears a consensus that Fe”-HRP is formed during the course of the reaction. Formation of a trace of Fe”-HRP as the initiation step provides the best explanation of our results. HRP-III formation from Fe”HRP and molecular oxygen was detected first by Yamazaki et al. (17) and confirmed by Wittenberg et al. (12). The partial inhibitory effect of SOD on HRP-III formation observed for the first time in the HRP/IAA system (Figs. 1B and 2B) indicates that there is more than one route for HRP-III formation, in which the following reaction occurs (18): HRP + 0; -+ HRP-III

[41

Superoxide could be formed by the sequence IAA’ + O2 * IAAO;

El P-31

IAAO’, + IAA+ + 0;

The rapid dissociation of a peroxyl radical to form superoxide can only be observed by pulse radiolysis experiments (19).

0.06

0.04

0.02

5,"

500

Wavelength

FIG. 9. Time course of spectral changes observed in the reaction native HRP, b, 20 s; c and d, 12 and 60 min after IAA addition.

550

600

650

7w

0.00

(nm)

of HRP (4.6 PM) with IAA at pH 7.0 A, 50

pM

IAA, B, 0.5 mM IAA. Scan a,

32

METODIEWA

ET AL.

The IAA/HRP

Reaction in the Presence of Hz02

In this paper we also have presented the first rapidscan spectral analysis of the initial conversion of HRP in the presence of Hz02 and IAA (Figs. 10 and 11). The results indicate that at pH 5.0 in the presence of H202, HRP oxidizes IAA as follows: 0.00L .3so

380

4cQ

420

FIG. 10. Rapid-scan spectra with HzOz (5.0 PM) (A) or with A, scans a-f-10,15,20,25,80, HzOz. B, scans a-d-20, 25,40, HzOz and IAA.

I, 440360 380 Wavelength (nm)

400

of the reaction of native H202 and IAA (2.5 mM) and 830 ms after mixing and 80 ms after mixing

420

440

I

HRP (0.8 pM) (B) at pH 5.0. of HRP with of HRP with

HRP + HzOz --* HRP-I + Hz0

PI

HRP-I + IAA * HRP-II + IAA’

PI [lOI WI

HRP-II + IAA + HRP + IAA’ HRP-II + H202 * HRP-III or

The inhibitory effect of histidine shown in Fig. 3 indicates a role for singlet oxygen (20) which has been claimed to be formed by the bimolecular reaction of IAAOH radicals (21, 22). Figure 4 shows that the catalytically inactive forms P670 and P-630 are enzymatic products of the reaction of HRP with IAA at pH 5.0 (23, 24). The results in Figs. 5 and 6 indicate that a precursor of P-670 may be formed (25). The results in Figs. 5 and 6 do not confirm the mechanism proposed by Smith et al. involving Fe”-HRP and 05 formation (4). The accelerated decay of HRP-III in the presence of IAA (Figs. 5-7) clearly indicates that IAA is a substrate for HRP-III and the reaction converts it to P-670. This result also indicates the formation of IAA’ and the possible propagation of chain reactions (22). Changes Observed at pH 7.0

There are significant changes in the reaction between native HRP and IAA at pH 7.0 compared to pH 5.0 (19, 29). The reaction is about 10 times slower at pH 7.0 (compare Figs. 1,2, and 8). The intermediate species (absorption maxima at 420 nm, Fig. 8) could result from the binding of IAA to the low-spin ferrous form of HRP to form an Fe”-HRP - - * IAA complex (27). The formation of stable HRP-II enhanced in the presence of SOD supports the ferrous character of the observed intermediate and formation of HRP-II in the reaction (28) Fe”-HRP + HzOz + HRP-II + Hz0

HRP + IAA + HRP-III

WI

The classical peroxidase cycle results in HRP-III formation, which is insensitive to SOD. This clearly indicates that superoxide does not serve as an active intermediate in the enzyme conversion. At pH 7.0 one can only observe HRP-II formation; HRP-III is not formed. SOD enhances the formation of HRP-II perhaps by preventing the reaction (31) HRP-II + 05 + HRP + Oz

1131

Conclusions

It is clear that the mechanism of IAA oxidation by HRP depends upon the amount of HzOz in the system. The primary product of the IAA donor molecule in oxidation or peroxidation catalyzed by HRP is a free radical (IAN). Therefore, the possibilities for propagation of chain reactions (8,9,22), independent of the initial step and leading to enzyme inactivation, should be considered. HRP is known to be one of the most stable enzymes. Nevertheless, in its reaction with IAA, it is transformed via Fe”HRP and HRP-III to the catalytically inactive forms P-

[71

The H202 results from the d&mutation of 0;. We do not have an explanation for the enhanced P-670 formation caused by SOD. The reaction of HRP-III with several electron donors is pH-independent (29), but it has been suggested that among all known hydrogen donors, IAA seems to be a peculiar substrate (30). It can be seen that a change in pH changes reaction rates and probably also the reaction mechanism.

0.03’

L

400 420 Wavelength (nm)

I 440

FIG. 11. Rapid-scan spectra of reaction of HRP-I with IAA at pH 5.0. IAA, 0.25 and 50 mM citrate buffer. Scan a, HRP-I only; scans bh-10, 15, 20, 30, 40, 50, and 80 ms after mixing.

OPTICAL

SPECTRA

OF PEROXIDASE/INDOLEACETIC

ACID

33

REACTION

670 and P-630 as final products. The free radicals IAK are scavenged by molecular oxygen to form peroxyl radicals IAAO’, . The oxygen uptake and chemiluminescence studies reported in the following paper shed further light on the nature of the peroxyl radicals and their role in the mechanism of reaction of IAA with HRP.

12. Wittenberg, J. B., Noble, R. W., Wittenberg, B. A., Antonini, E., Brunori, M., and Wyman, J. (1967) J. Biol. Chem. 242,626-634.

ACKNOWLEDGMENTS

16. Ricard, J., and Nari, J. (1967) Biochim.

13. Ohlsson, 373-375.

P.-I., and Paul, K.-G.

(1976) Acta Chem. &and.

B30,

14. Briggs, R. G., and Fee, J. A. (1978) Biochim. Biophys. Acta 537, 86-99. 15. Yamazaki, H., and Yamazaki, I. (1973) Arch. Biochem. Biophys.

154,147-159. Research by the authors at the University of Alberta was supported by the Natural Sciences and Engineering Research Council of Canada. Financial support for the study leave of H.B.D. at the University of SHo Paul0 by Banco International de Desenvolvimento at U.S.P. is gratefully acknowledged.

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