©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Role of Arginine 38 in Horseradish Peroxidase
A CRITICAL RESIDUE FOR SUBSTRATE BINDING AND CATALYSIS (*)

(Received for publication, August 22, 1995; and in revised form, November 21, 1995)

Jose Neptuno Rodriguez-Lopez (1)(§) Andrew T. Smith (2) Roger N. F. Thorneley (1)(¶)

From the  (1)From Nitrogen Fixation Laboratory, Joseph Chatt Building, John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH, United Kingdom and the (2)Department of Biochemistry, University of Sussex, Brighton, BN1 9QG, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The observed pseudo-first order rate constant for the reaction between a horseradish peroxidase (HRP) variant (R38L)HRPC and hydrogen peroxide saturates at high peroxide concentrations (K = 11.8 mM). The data are consistent with a two-step mechanism involving the formation of an HRP-H(2)O(2) intermediate (k = 1.1 times 10^4M s) whose conversion to compound I is rate-limiting (k = 142 s) suggesting that Arg-38 is not only involved in the cleavage of the O-O bond of peroxide but also has an important role in facilitating the rapid binding of H(2)O(2) to HRP. Rapid-scan spectrophotometry revealed the presence of a transient intermediate with a spectrum consistent with a ferric-hydroperoxy complex. At high peroxide concentrations (>500 µM), compound I is converted to compound III without the accumulation of compound II. Spectrophotometric titrations show that arginine 38 is also involved in modulating the apparent affinity of HRPC for reducing substrates such as guaiacol and p-cresol. The spectrum of the complex formed when these substrates bind to the ferric form of the mutant enzyme differs from that observed when they bind to the wild-type ferric enzyme. At neutral and alkaline pH compound I of (R38L)HRPC was stable and reduced to ferric enzyme without apparent formation of compound II upon titration with p-cresol or ascorbic acid, suggesting a change in the rate-limiting step in the peroxidase cycle. Steady-state kinetic analyses carried out at pH 7.0 showed significant increases in the apparent K for guaiacol, p-cresol, and 2,2`-azinobis(3-ethylbenzothiazolinesulfonic acid) (ABTS). The high stability of the oxyferryl form of (R38L)HRPC and its low catalytic constant for reducing substrates also shows that arginine 38 modulates the reactivity of HRP compound I.


INTRODUCTION

Peroxidases are enzymes catalyzing the oxidation of a variety of organic and inorganic compounds by hydrogen peroxide or related compounds. Horseradish peroxidase (HRP) (^1)has a carbohydrate content of 18% (1) and consists of more than 30 multiforms(2) . The predominant form is isoenzyme C (HRPC) that is a monomeric glycoprotein of 308 residues with eight oligosaccharide side chains. It has a calculated protein M(r) of 33,922 (3) and has been shown to contain up to 2 mol of calcium ions/mol of protein(4) . Peroxidase action involves 2e oxidation of the enzyme by hydrogen peroxide to give a species known as compound I. This then reverts to the resting state via two successive 1e reactions with reducing substrate molecules, the first yielding a second enzyme intermediate, compound II(1) . Reduction of compound II to native enzyme is often rate-limiting in the peroxidase catalytic cycle. However, operating with limiting concentrations of H(2)O(2) and a large excess of reducing substrate it is possible to make compound I formation the rate-controlling step but it is impossible to make the conversion of compound I to compound II rate-limiting for native HRPC(1) .

Poulos and Kraut (5) proposed a mechanism for the peroxidase-catalyzed heterolytic cleavage of the RO-OH bond based on the crystal structure of cytochrome c peroxidase. Two essential features of this mechanism were acid-base catalysis by a distal histidine (His-42 in HRP) and charge stabilization of a precursor enzyme-substrate complex by the conserved distal arginine (Arg-38). Hence, compound I formation is proposed to be a two-step mechanism. In the first step there is a pre-equilibrium of reactants to form at least a precursor complex, HRP-H(2)O(2), and in the second step the O-O bond is cleaved producing compound I and water:

Kinetic evidence for this mechanism in HRPC (6, 7) and microperoxidase (8) has been obtained using stopped-flow cryoenzymology. The spectrum of a new intermediate, proposed to be an hyperporphyrin species (compound 0) was obtained at [H(2)O(2)] > K. It was proposed that this new intermediate was formed from an HRP-H(2)O(2) complex after an oxidation step involving electron transfer between HO(2) and the ferric heme group. Mechanism 1 has also been proposed for cytochrome c peroxidase following studies with cytochrome c peroxidase variants and hydrogen peroxide(9, 10) .

The proposed role of Arg-38 in the formation of compound I suggests that changing this polar residue to a neutral one should significantly decrease the stabilization of the precursor enzyme-substrate complex and inhibit the subsequent heterolytic cleavage of the oxygen-oxygen bond. In an attempt to observe the formation and accumulation of compound 0 we have constructed a HRP mutant in which Arg-38 has been replaced by Leu using site-directed mutagenesis. This HRPC variant has been characterized with respect to its reaction with hydrogen peroxide and to the reactivity of its compound I to different reducing substrates. Nuclear Overhauser (11) and NMR relaxation(12, 13) experiments suggest that phenol and other aromatic substrates bind distal to the heme and close to the heme C18 methyl with the aromatic protons 6-10 Å from the heme group(14) . Different binding sites for guaiacol, thioanisol, and iodide have been proposed based on studies of HRP modification with -meso substituents(15, 16) . However, the exact nature of the reducing substrate binding site of HRPC is still not known and the role of distal heme residues such as histidine 42 and arginine 38 not resolved(14) . Substitution of arginine 38 by leucine in HRPC gave us the opportunity to study the role of this residue in modulating the binding and activity of reducing substrates with the enzyme.


MATERIALS AND METHODS

Reagents

HRP (EC 1.11.1.7) isoenzyme C (Type VI) was purchased from Sigma and used without further purification. HRPC was obtained as described by Smith et al.(17) . The concentrations of HRPC and HRPC were determined spectrophotometrically by using = 102 mM cm(18) and = 92 mM cm, respectively. Reagent grade H(2)O(2) (30% v/v) was obtained from BDH and its concentration was calculated by iodide titration with HRPC(19) . ABTS, guaiacol, and p-cresol were purchased from Sigma and ascorbic acid from Merck. Stock solutions of the reducing substrates were prepared in 0.15 mM phosphoric acid to prevent autoxidation. All other chemicals were of analytical grade and supplied by Merck.

Construction of an (R38L)HRPCGene

A polymerase chain reaction-based technique with the HRPC synthetic gene (17) as template was used to generate insert DNA bearing the Arg-38 mutation. The construction of the (R38L)HRPC gene was carried out as described by Smith et al.(20) for the (F41V)HRPC mutant, except that oligonucleotide N1 replaced V1. N1 was identical to the wild-type synthetic gene sequence between nucleotide positions 111-147, except that it contained the Leu codon TTA at position 126 (5`-GCTGCTTCAATATTATTACTGCACTTCCATGAC-3`).

Amplified DNA fragments (404 base pairs) bearing this mutation were cloned in-frame into the wild-type gene at the unique SspI and XhoI sites in the HRP expression vector pAS5(20) . Double-stranded DNA sequence analysis by the dideoxy chain termination method, using Sequenase version 2 (21) and oligonucleotides S1 and N1 (20) as sequencing primer, confirmed the expected sequence change.

Preparation of (R38L)HRPC

Growth and induction of Escherichia coli strains producing the recombinant peroxidase variant were as described previously(20) . Folding and activation of (R38L)HRPC recovered from E. coli inclusion bodies were achieved essentially by the method of Smith et al.(17) with the modifications subsequently described by Smith et al.(20) . Purified enzyme was desalted into 10 mM sodium MOPS, pH 7.0, and stored in liquid nitrogen until use. The concentration of the enzyme was determined using the Soret extinction coefficient determined by the pyridine hemeochrome method (22) .

Pre-Steady State Kinetics

Transient kinetics were monitored in a stopped-flow spectrophotometer (model SF-51, Hi-Tech Scientific, Salisbury, UK) in 10 mM sodium phosphate buffer, pH 7.0. Data were recorded through an RS232 interface with a microcomputer. Compound I formation was monitored at 401 nm, isosbestic for compound I and compound III (see ``Results''). (R38L)HRPC compound I was generated by mixing enzyme (2.0 µM) with 2 volumes of hydrogen peroxide (4.0 µM) in a simple flow-mixing device and was used within 5 min of preparation. Temperature was controlled at 25 or 10 °C, using a Techne C-400 circulating bath with a heater-cooler.

Spectrophotometry

Stopped-flow rapid-scan spectrophotometry was carried out with the same stopped-flow spectrophotometer described above equipped with an MG 6000 diode array system. Ultraviolet/visible absorption spectra were recorded in quartz cuvettes (1 cm) on a Shimadzu UV-2101PC spectrophotometer with a spectral bandwidth of 1 nm and a scan speed of 120 nm/min. Steady-state assays were carried out with the same instrument by measuring the appearance of products. Tetraguaiacol formation was followed at 470 nm ( = 26.6 mM cm), 4-methyl-o-benzoquinone at 400 nm ( = 1.14 mM cm), and ABTS radical at 414 nm ( = 31.1 mM cm). The assay medium was 10 mM sodium/phosphate buffer, pH 7.0. Other conditions and reagents are detailed in the text and in the legends to figures and tables.

Spectrophotometric Determination of Substrate Binding Constants

Difference spectra of the Soret region (350-480 nm) of the ferric (R38L)HRPC with reducing substrates minus ferric (R38L)HRPC were recorded in 1-cm quartz microcuvettes in 10 mM sodium MOPS, pH 7.0. The reference and the sample cuvettes contained 0.4 ml of enzyme solution (10 µM) for base line recording. Increasing amounts of guaiacol (in the same buffer as the enzyme) or p-cresol (in ethanol) were then added to the sample cuvette. The same volume of solvent was added to the reference cuvette. The contents were stirred with a plastic rod before recording the spectrum. Equilibrium dissociation constants (K(d)) for the complex formation were calculated using the following expression(12) :

where DeltaA is the change in absorbance, S is the substrate concentration, and DeltaA is the change in absorbance at saturating concentration of the substrate. Similar titrations were carried out with plant and recombinant HRPC. In all cases the K(d) values were 3 orders of magnitude greater than the enzyme concentration and the free concentration of substrate was assumed to be equal to its initial concentration.

Kinetic Data Analysis

Pre-steady state kinetic data were analyzed by fitting the absorbance time curves to exponential functions using a least-squares minimization procedure. The values of K(m) and V(max) for HRPC and (R38L)HRPC on varying H(2)O(2) at fixed saturating concentrations of reducing substrate were calculated by triplicate measurements of at each [H(2)O(2)] concentration. The same procedure was used to determine these kinetic constants for reducing substrates using a saturating concentration of H(2)O(2). The reciprocal of the variances of were used as weighting factors in the non-linear regression fitting of versus [substrate] data to the Michaelis-Menten equation(23) . The fitting was carried out by using a Gauss-Newton algorithm (24) implemented in a BASIC program. Initial estimates of K(m) and V(max) were obtained from the Lineweaver-Burk equation.


RESULTS

Properties of (R38L)HRPC

The enzyme, which was purified from the folding medium by FPLC using cation-exchange chromatography on a Mono-S HR5/5 column (Pharmacia), eluted at the same salt concentration as HRPC. Preparations were judged to be homogeneous by the observation of a single band on a Coomassie Blue-stained reducing SDS-polyacrylamide gel electrophoresis gel. The spectrum of (R38L)HRPC at pH 7.0 is shown in Fig. 1. This enzyme has a similar spectrum to that reported for the same variant in cytochrome c peroxidase (9) but with a 3-nm displacement of the Soret band to 399 nm in the HRP mutant. Moreover, the spectrum of (R38L)HRPC has peaks at 500 and 646 nm and a shoulder at 383 nm. The extinction coefficient for the Soret maximum (399 nm) calculated using the pyridine hemeochrome method was 86 mM cm. The preparations used had ratios of absorbance at 398/280 nm (Rz value) of 3.2. Heme incorporation data indicated that the preparations were not contaminated with inactive heme-free enzyme (100% heme incorporation). The electronic absorption spectrum of (R38L)HRPC* in the Soret region is unusually broad and although the spectrum appears consistent with pentacoordination of the heme iron at pH 7.0 more conclusive resonance Raman data are required for a precise determination of the heme coordination state of this mutant(25) .


Figure 1: Spectrum of (R38L)HRPC (solid line) and (R38L)HRPC compound I (dashed line) in 10 mM Na-phosphate buffer, pH 7.0. Compound I was formed as was described under ``Materials and Methods.''



Reaction of (R38L)HRPCwith Hydrogen Peroxide

(R38L)HRPC reacted with a 10-fold excess of hydrogen peroxide to slowly yield a stable compound I species (Fig. 2). The spectrum for this intermediate was exactly the same as that described for native HRPC compound I (18) with an extinction coefficient of 57 mM cm in the Soret region. (R38L)HRPC compound I was stable in the reaction media for more than 30 min. After this time, compound I slowly converted to the ferric state with no detectable accumulation of compound II (data not shown). In contrast when the same experiment was carried out with wild-type HRPC, a very unstable compound I was formed which converted spontaneously to compound II with an isosbestic point at 395 nm. Compound II was subsequently reduced to resting ferric enzyme.


Figure 2: Rapid-scan stopped-flow of the reaction of (R38L)HRPC (1.6 µM) with a 10-fold excess of hydrogen peroxide (16 µM) in 10 mM Na-phosphate buffer, pH 7.0, 25 °C. The first scan was taken 4.4 ms after the flow stopped, and the subsequent scans were at 0.5-s intervals. The arrows show the direction of absorbance change with time.



In order to determine the pseudo-first order rate constant for compound I formation, k(a), the concentration of H(2)O(2) was increased through the range 20 µM to 50 mM. At high peroxide concentrations (>0.1 mM), compound I was formed over a period of seconds followed by compound III formation with an isosbestic point at 401 nm but without detectable accumulation of compound II (Fig. 3). This wavelength was used for the determination of k(a). The time dependences of the absorbance change at 401 nm can be fitted to a simple exponential function. Under pseudo-first order conditions, with hydrogen peroxide in large excess, the dependence of k(a) was linear at low hydrogen peroxide concentration (leq0.5 mM) (Fig. 4A), whereas at high hydrogen peroxide concentrations (up to 50 mM), k(a) approaches a limiting value (Fig. 4B). These dependences can be explained if the reaction between (R38L)HRPC and H(2)O(2) follows the mechanism described in . If the first step in this mechanism equilibrates rapidly (k(1)[H(2)O(2)] + k approx k(2)) the expression for k(a) is:


Figure 3: Rapid-scan stopped-flow of the formation of (R38L)HRPC compound I and its conversion to compound III in 10 mM Na-phosphate buffer, pH 7.0, 25 °C. The reaction was started by mixing 2 µM enzyme and 0.5 mM hydrogen peroxide. The first scan was taken 1.23 ms after the flow stopped, and the subsequent scans were at 1-s intervals. The arrows indicate the direction of absorbance change with time.




Figure 4: Dependence of k, the pseudo-first order rate constant for (R38L)HRPC compound I formation, on the hydrogen peroxide concentration at pH 7.0 and 25 °C. A, 20-500 µM hydrogen peroxide. B, 0.02-50 mM hydrogen peroxide.



where k = k and K = (k + k)/k(26) . Therefore, k is directly calculated from the limiting value (Fig. 4B), whereas k can be determined assuming that k k from the slope of the dependence of kversus [HO] at low concentrations of hydrogen peroxide (Fig. 4A). The values for the elementary rate constants for (R38L)HRPC compound I formation are k = 1.1 ± 0.1 times 10M s and k = 142 ± 10 s. The value of k is about 3 orders of magnitude lower than that for native HRPC (1.7 ± 0.1 times 10M s) and non-glycosylated recombinant HRPC (1.6 ± 0.1 times 10M s).

Direct Observation of the HRP-H(2)O(2)Intermediate

The saturation kinetics observed with (R38L)HRPC indicate that at high [H(2)O(2)] a reaction intermediate is formed in a rapid pre-equilibrium step prior to compound I formation. Simulation of mechanism 1 using the measured rate constants for the (R38L)HRPC mutant shows that at 50 mM hydrogen peroxide the time required for the maximum accumulation of the intermediate is 2.5 ms. In order to minimize any possible stopped-flow artifacts occurring in the first few milliseconds of the reaction, the detection of this intermediate was carried out at a lower temperature. At 10 °C, the values for k(1) and k(2) were 6.3 times 10^3M s and 33 s, respectively, and the maximum concentration of the intermediate, 78% of the initial enzyme, was calculated to be formed at 10 ms. Under these experimental conditions, the resting enzyme converts to compound I but without the appearance of an isosbestic point, suggesting the accumulation of at least one intermediate, possibly the complex HRP-H(2)O(2). The time course at 374 nm, isosbestic between ferric enzyme and the transient intermediate, shows a lag period of about 6 ms (Fig. 5A). The solid circles are simulated data points calculated using and the above values of k(1) and k(2). The time course of the reaction (monitored at 357 nm isosbestic between ferric enzyme and compound I) exhibits a maximum absorbance at about 10 ms after which time the formation of an intermediate species is essentially complete and its conversion to compound I is in progress (Fig. 5B). Again the simulated data points shown in Fig. 5B lie on the observed absorbance time curve. The spectrum for this intermediate, shown in Fig. 6, resembles that of resting enzyme but with peaks at 397, 487, and 580 nm and a shoulder at 530 nm. This spectrum does not resemble that of the hyperporphyrin published by Baeck and Van Wart(6, 7) . We suggest that this intermediate is the HRP-H(2)O(2) complex. However, these data do not exclude the possibility of the subsequent formation of compound 0, although we were not able to detect directly this species under our conditions.


Figure 5: Time course at 375 nm (A) and 357 nm (B) of the reaction of 2 µM (R38L)HRPC with 50 mM H(2)O(2) in 10 mM Na-phosphate buffer, pH 7.0, 10 °C. The filled circles are simulated data points using a computer program KSIM (supplied by Dr. N. Millar). The rate constants used for the simulation, assuming mechanism 1, were k(1) = 6.3 times 10^3M s; k = 0.07 s and k(2) = 33 s, and the extinction coefficients used were: A, =




Figure 6: Rapid-scan optical spectra of the reaction of 2 µM (R38L)HRPC with 50 mM H(2)O(2) in 10 mM Na-phosphate buffer, pH 7.0, 10 °C. Curve a is the spectrum of (R38L)HRPC. Curves b and c are 10-ms and 0.1-s scans, respectively, initiated at the start of mixing, which show the formation of the HRP-H(2)O(2) complex and its conversion to compound I.



Binding of Reducing Substrates to Ferric (R38L)HRPC

Spectrophotometric titrations have shown that substrates can be divided into two classes on the basis of the difference spectra obtained when they bind to HRP(16, 27) . A type I spectrum is induced by p-cresol while guaiacol gives a type II spectrum. The difference spectrum obtained when guaiacol binds to plant HRPC (data not shown), and the K(d) value (Table 1), determined from the substrate concentration dependence of the amplitude of the difference spectra, were essentially identical to those previously reported(27) . Similar results were obtained when guaiacol bound to recombinant HRPC (Fig. 7; Table 1). However, the difference spectrum caused by binding of guaiacol to (R38L)HRPC was quite different (Fig. 7) and resembles the type I spectra reported for the binding of phenol, hydroquinone, and p-cresol to ferric HRPC(27) . The spectrum had a trough with a minimum at 395 nm, a peak at 416 nm, and an isosbestic point at 406 nm (Fig. 7). The value of K(d) for the binding of guaiacol to (R38L)HRPC showed that it bound 2.3 times weaker than it did to both plant and recombinant HRPC (Table 1).




Figure 7: Difference spectrum for the binding of guaiacol (A) and p-cresol (B) to HRPC, and guaiacol (C) and p-cresol (D) to (R38L)HRPC.



Spectrophotometric studies of the binding of p-cresol to (R38L)HRPC also showed that the mutation modified the binding of this substrate to the enzyme. Both HRPC (28) and HRPC (Fig. 7) gave a type I spectrum and the calculated values for K(d) (Table 1) were very similar to those reported for the native enzyme(28) . Binding of p-cresol to ferric (R38L)HRPC induced a spectrum with peaks at 385 and 413 nm (Fig. 7). The value of K(d) for the binding of p-cresol to ferric mutant enzyme was about 2.5 times higher than for the binding of this substrate to both wild-type enzymes (Table 1). Therefore, substitution of the distal arginine 38 by leucine in HRPC differentially modifies both the equilibrium constant for the binding of aromatic electron donors to the ferric state of the enzyme and the coordination state of the complex formed between ferric HRP and aromatic substrates.

Reaction of (R38L)HRPC Compound I with Reducing Substrates

When (R38L)HRPC compound I was kinetically titrated with p-cresol or ascorbate, it was apparently reduced directly to ferric enzyme with no accumulation of compound II. It is well known that compound II exists in two pH-dependent forms (pK = 8.6), the protonated form being more reactive than the unprotonated form(1, 29) . Titration experiments with (R38L)HRPC compound I at pH 10, designed to stabilize compound II, again failed to detect any compound II (Fig. 8). This was similar to the behavior reported for (R38K)HRPC(32) . Three different explanations are possible for this observation: (a) a novel reaction pathway exists; (b) compounds I and II are spectroscopically indistinguishable (cf. compounds I and II in cytochrome c peroxidase); or (c) the reduction of compound II to resting ferric enzyme is very much faster that the reduction of compound I at both high and low pH values.


Figure 8: The reduction of (R38L)HRPC compound I at pH 10 (10 mM Na borate buffer). Spectra, a, 2 µM (R38L)HRPC; b, 20 µM H(2)O(2) added to a; c, 1 µM ascorbic acid added to b; d, another 1 µM ascorbic acid added to c.



Steady-State Kinetics of (R38L)HRPCwith Reducing Substrates

The kinetic parameters for (R38L)HRPC acting on ABTS, guaiacol, and p-cresol and a comparison with the values obtained for the native plant and recombinant HRPC enzymes are given in Table 2. The Arg-38 mutation differentially increased the K(m) for hydrogen peroxide in the presence of these three substrates. The difference in the apparent value for K(m) at pH 5.0 between HRPC and HRPC was previously reported(20) . It was proposed that this difference could be related to the lack of glycosylation of the recombinant enzyme, allowing readier access of the bulky charged substrate ABTS to its binding site. The failure to observe the same effect on the value of K(m) for small substrates such as guaiacol and p-cresol supports this hypothesis. Therefore, comparing the value of K(m) for ABTS and guaiacol for the two non-glycosylated enzymes, HRPC and (R38L)HRPC, it is apparent that the effect of the mutation on the K(m) for both electron donors is essentially the same (Table 2). However, the effect of this mutation on the apparent K(m) for p-cresol was higher. The catalytic constant for the oxidation of ABTS by the recombinant wild-type enzyme is drastically decreased at pH 7.0 compared to the value at pH 5.0. This results in guaiacol and p-cresol being better reducing substrates at pH 7.0. However, the R38L mutation reverses this trend, with ABTS being a better substrate than guaiacol and p-cresol at pH 7.0. These results show that arginine 38 not only modulates the formation of compound I (increase in the K




DISCUSSION

Mechanism of Compound I Formation: Effects of Arg-38 Mutation

A mechanism for compound I formation must be able to explain the saturation kinetics observed at high hydrogen peroxide concentration and be consistent with the formation of an enzyme-substrate complex whose conversion to compound I is rate-limiting for reactions in the absence of reducing substrates. The simplest mechanism that is consistent with the data is mechanism 1. The initial second-order step with implicit deprotonation of the peroxide molecule is the binding of the anionic HO(2) ligand to the Fe(III) with proton transfer to a basic amino acid residue. This is followed by a first-order step in which the oxygen-oxygen bond is cleaved to yield compound I and water. However, a more complex mechanism, needed to explain the formation of other intermediates (i.e. compound 0; (6, 7, 8) ), is also compatible with our data, although under our conditions we did not detect any intermediates with a spectrum similar to that proposed for compound 0.

Substitution of arginine 38 by Leu in HRP modifies both the second-order rate constant of the formation of HRP-H(2)O(2) and the first-order rate constant for subsequent compound I formation. The mutation decreases by 3 orders of magnitude the first rate constant but it is not easy to estimate by how much it decreases the second. It has been proposed that the O-O cleavage step is very fast for wild-type peroxidases, probably of the order of 10^5 s at room temperature(9, 30) . If this value is true the decrease induced by the arginine to leucine mutation would also be about 3 orders of magnitude. However, we believe that this value is not consistent with previous data. A value of 10^5 s for k(2) would mean that the value of the K

Smulevich et al.(25) have recently discussed the effect of changing Arg-38 Lys in HRP and Arg-48 Lys in cytochrome c peroxidase. The cytochrome c peroxidase variants R48K and R48L react with hydrogen peroxide to form compound I factors of 2 and 200 times slower, respectively, than does native cytochrome c peroxidase(31) . In contrast, the R38K variant in HRPC showed a 500-fold decrease in the rate of compound I formation(32) . Therefore, in cytochrome c peroxidase a lysine residue is able to substitute quite well for arginine 48 with respect to compound I formation, but it is not able to do so nearly as efficiently in HRP. The data presented above show that the Arg Leu mutation decreases the second-order rate constant for compound I formation by 1,200-fold and k(2) by 10-100 times. This is not only consistent with the Poulos-Kraut mechanism (5) but also indicates that arginine 38 plays an important role in the binding step of the peroxide molecule in the distal heme pocket as previously suggested for an HRPC mutant in which arginine 38 had been replaced by lysine(32) . Two possible functions of arginine 38 in hydrogen peroxide binding are that the polar character of this residue facilitates the access of the hydrogen peroxide molecule to the heme and/or provides an electrostatic interaction with the incoming peroxide which may also induce the deprotonation of hydrogen peroxide at neutral pH.

The effect of Arg-38 mutations in HRPC on the binding constant could explain the low activity of metmyoglobin with respect to hydrogen peroxide (k(1) = 10^2M s)(33) . In metmyoglobin the distal histidine is still present in the active site, but Phe-43 takes the place of the distal arginine(34) . The presence of a phenylalanine residue in the active site could also have a profound steric effect on the accessibility of hydrogen peroxide to the distal pocket thereby decreasing the binding constant by up to 5 orders of magnitude. However, the replacement of His-42 by Leu in HRPC decreases the binding constant for hydrogen peroxide by 5 orders of magnitude. (^2)This suggests that the principal feature in the formation of an activated enzyme-HO(2) complex is the transfer of a proton to the distal histidine to form an imidazolium side chain thereby promoting the binding of HO(2) to the heme. It seems likely that both events take place in a concerted manner involving both the catalytic arginine and histidine. The effect of the Arg-38 Leu mutation on k(2) is most likely due to the proposed role of this residue in stabilizing the transition state during O-O cleavage to form compound I and water(5) .

Possible Structure for the ES Complex

The optical spectrum of the newly detected intermediate provides some information as to its likely structure. The spectrum of the intermediate is not very different from that of the ferric enzyme. This is not surprising since the (TMP)Fe(III)(t-BuO(2)) complex and other ferric-oxyanion heme complexes have relatively normal Soret- and Q-band spectra(35, 36) . The spectrum for this species shows the loss of the 380 nm shoulder and small shifts in the 505- and 645-nm bands. Moreover, an increase in the Soret extinction coefficient is also predicted. Having noted the similarity of these changes to those observed when cytochrome c peroxidase ages(37) , we suggest that this new intermediate is a ferric hydroperoxy complex in which one oxygen atom of HO(2) is coordinated to the iron atom. Recently, a 2.2-Å crystal structure of cytochrome c peroxidase oxyperoxidase has been obtained and considered as a model for the transient ferric enzyme-peroxide complex(38) . The authors support their proposal by arguing that because oxyperoxidase and the enzyme-hydrogen peroxide complex differ by only one electron and one proton their structures are likely to be very similar. However, we did not detect any species with a spectrum similar to that reported for oxyperoxidase in the reaction of (R38L)HRPC with hydrogen peroxide. On the other hand, it is important to note that the compound I and compound II forms of both plant and fungal peroxidases that differ by only one electron have completely different absorption spectra.

The Aromatic Donor Molecule Binding Site of HRP

The non-availability of a high resolution crystal structure of HRP makes the identification of the binding site(s) for aromatic donor molecules difficult. NMR data (12, 13) and enzyme inactivation studies(15, 16) have located the aromatic substrate binding site near the -meso-heme edge. The side chains of two phenylalanine residues are also implicated in the binding process(14, 39) . The possibility of two different binding sites, one for guaiacol or resorcinol, that induce type II spectra when they bind to HRPC, and a second site for phenols such as p-cresol, hydroquinone, and aniline, that yield a type I spectrum, has been ruled out by NMR relaxation studies(12, 13) . It has been concluded that the UV/visible spectroscopic differences reflect different hydrogen bonding interactions to residues that modulate the chromophore rather than binding to different sites. The direct participation of distal heme residues such as histidine 42 and arginine 38 in the binding of aromatic electron donors remains unclear. Thus, histidine 42 has been proposed to be involved in the binding of aromatic substrates(14) , however, chemical modification of HRP with diethyl pyrocarbonate, a histidine-specific reagent, did not modify the dissociation constant for guaiacol binding(40) . However, it has been reported that replacement of arginine 38 by lysine in HRPC greatly decreased the affinity for benzhydroxamic acid(32) . Additional evidence for arginine 38 being directly involved in the binding of aromatic substrates is presented in this paper. Substitution of arginine 38 by leucine increases the dissociation constants for the binding of guaiacol and p-cresol to the ferric enzyme, possibly by modifying the hydrogen bond interactions in the complexes formed when they bind to the enzyme (Fig. 7). This is consistent with the effect on the binding of these reducing substrates to compound I and/or compound II as indicated by the increase in the apparent K(m) values. These large differences in affinity cannot be rationalized simply on the basis of a local perturbation resulting from a single-site substitution. An arginine residue in a protein often serves as a cationic site for the binding of a negatively charged group in a substrate or cofactor(41) . Hence the simplest explanation for the role of arginine 38 in substrate binding is a direct electrostatic interaction between the positively charged guanidinium group of arginine 38 and the partial negative charge developed on the oxygen of the phenolic group of substrates such as p-cresol and guaiacol, causing the orientation of the substrate in the active site prior to electron transfer or hydrogen atom transfer in a similar way to that proposed for the binding of benzhydroxamic acid(32) . The polar character of arginine 38 may also facilitate the access of the reducing substrate to its binding site on compound I. These results, therefore, support models based largely on NMR data that indicate key interactions between bound substrates and the heme methyl C^18H(3), the conserved residue arginine 38 and also possibly histidine 42(12, 13, 14) .

The Rate-limiting Step in the Peroxidase Cycle

One possible explanation for the failure to observe compound II in the reaction of (R38L)HRPC compound I with reducing substrates is that an increase in the stability of compound I with respect to compound II is induced by the mutation. It is well established that the reduction of compound II to native enzyme is usually the rate-limiting reaction in the peroxidase cycle. The rates of reduction of compound I and II often differ by factors in the range of 20-100(42) . However, Arthromyces ramosus peroxidase (43) forms a very stable compound I while compound II is unusually unstable being rapidly reduced to ferric enzyme. The instability of A. ramosus peroxidase compound II was explained in terms of a higher reduction potential for this species. A similar situation is observed in (R38L)HRPC. The drastically reduced reactivity of the compound I formed with a 10-fold excess of hydrogen peroxide supports the hypothesis that the reduction of compound II is not rate-limiting for this variant. However, the failure to detect compound II at pH 10 causes us not to completely exclude the possibility of novel reduction pathways for compound I and/or compound II.

Mechanism of Compound I Reduction in HRP: Role of Arg-38

The data presented above indicate that arginine 38 is not only involved in compound I formation but also in other partial reactions of the catalytic cycle. Substitution by leucine renders compound II undetectable and modifies the kinetic constants for the reactions with reducing substrates. As discussed above, the modification of the apparent K(m) values for guaiacol, p-cresol, and ABTS suggests that Arg-38 also has a role in the binding/orientation of electron donors in the active site of HRPC compound I. However, this effect alone cannot explain the decrease in k, since the rate equation for k does not contain a binding constant term. Other systems in which an arginine residue influences catalysis by electrostatic effects include 2-keto-4-hydroxyglutarate aldolase (44) and transketolase(45) . We suggest that arginine 38 could have a catalytic role in the reduction of HRP compound I. Interaction between the guanidinium group of arginine 38 and the ferryl oxygen of HRP compound I would be expected to withdraw electron density from the porphyrin radical cation and, thus facilitate the electron transfer from the reducing substrate by increasing the redox potential. This function of arginine 38 could explain both the decrease in the k for electron donors and the different effect of this substitution in the reactivity of cytochrome c peroxidase (9) where the radical cation is not located on the porphyring ring, but on Trp-191 which is remote from the proposed substrate binding site and arginine 48(46, 47) .

The differential effect of the (R38L) mutation on HRP and (R48L)cytochrome c peroxidase reactivities can, in part, be explained in terms of the different polarity of their respective distal heme pockets. Thus, the more polar pocket in cytochrome c peroxidase can still effectively promote charge separation in the absence of the positively charged arginine. On the other hand, our data clearly show that the removal of a positive charge in the active site of HRP makes the reactivity for hydrogen peroxide similar to that reported for metmyoglobin. In addition, we have also recently studied by laser photolysis the reaction of the ferrous form of (R38L)HRPC with other heme ligands such as carbon monoxide and cyanide ion(48) . Removal of arginine 38 from the active center of HRP differentially modulates the kinetics of binding of CO and HCN to the heme. The reassociation rates for both ligands approach those previously reported for sperm whale myoglobin and human hemeoglobin (49, 50) . Engineering proteins with oxygen binding properties from peroxidases is now a real possibility.


FOOTNOTES

*
This work was supported in part by a grant from the European Communities Human Capital and Mobility Programme ``Peroxidase in Agriculture and in the Environment'' (contract ref. ERB CHRX-CT92-0012). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a fellowship from the European Communities as part of the above programme.

To whom correspondence should be addressed: Nitrogen Fixation Laboratory, Joseph Chatt Bldg., John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK. Tel.: 01603-456900 (ext. 2739); Fax: 01603-454970.

(^1)
The abbreviations used are: HRPC, horseradish peroxidase isoenzyme C (glycosylated plant enzyme); HRPC, non-glycosylated recombinant horseradish peroxidase; (R38L)HRPC, Arg-38 Leu HRPC mutant; (R38K)HRPC, Arg-38 Lys HRPC mutant; (F41V)HRPC, Phe-41 Val HRPC mutant; ABTS, 2,2`-azinobis(3-ethylbenzothiazolinesulfonic acid); MOPS, 3-morpholinopropanesulfonic acid.

(^2)
J. N. Rodriguez-Lopez, A. T. Smith, and R. N. F. Thorneley, unpublished data.


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