Role of Arg112 of cytochrome p450cam in the electron transfer from reduced putidaredoxin. Analyses with site-directed mutants.

The mechanism for the reduction of ferric cytochrome P450cam by reduced putidaredoxin, the physiological electron donor for the cytochrome, has been studied by using site-directed mutants of cytochrome P450cam, in which Arg112, an amino acid residue at the presumed binding site for putidaredoxin, was changed to several other amino acid residues. The affinity of reduced putidaredoxin for ferric cytochrome P450cam to form a diprotein complex was decreased greatly by changing Arg112 to a neutral amino acid such as Cys, Met, or Tyr. The rate of intracomplex electron transfer from putidaredoxin to cytochrome P450cam also diminished upon replacing the basic residue with neutral ones, being 42, 18, 4.0, 1.3, and 0.16 s−1 for Arg (wild type), Lys, Cys, Met, and Tyr enzymes, respectively. Furthermore, the oxidation-reduction potential of cytochrome P450cam (Fe3+/Fe2+ couple) decreased in a similar way to the decrease in the rate of electron transfer upon amino acid substitution; the values were −138, −162, −182, −200, and −195 mV for Arg (wild type), Lys, Cys, Met, and Tyr enzymes, respectively. These results indicate that the amino acid substitution at position 112 affects the oxidation-reduction potential of the heme iron in cytochrome P450cam, thereby diminishing the rate of electron transfer between the two metal centers. The rate of electron transfer from putidaredoxin to oxyferrous cytochrome P450cam also diminished upon substitution of Arg112 with a neutral amino acid.

The mechanism for the reduction of ferric cytochrome P450 cam by reduced putidaredoxin, the physiological electron donor for the cytochrome, has been studied by using site-directed mutants of cytochrome P450 cam , in which Arg 112 , an amino acid residue at the presumed binding site for putidaredoxin, was changed to several other amino acid residues. The affinity of reduced putidaredoxin for ferric cytochrome P450 cam to form a diprotein complex was decreased greatly by changing Arg 112 to a neutral amino acid such as Cys, Met, or Tyr. The rate of intracomplex electron transfer from putidaredoxin to cytochrome P450 cam also diminished upon replacing the basic residue with neutral ones, being 42, 18, 4.0, 1.3, and 0.16 s ؊1 for Arg (wild type), Lys, Cys, Met, and Tyr enzymes, respectively. Furthermore, the oxidation-reduction potential of cytochrome P450 cam (Fe 3؉ / Fe 2؉ couple) decreased in a similar way to the decrease in the rate of electron transfer upon amino acid substitution; the values were ؊138, ؊162, ؊182, ؊200, and ؊195 mV for Arg (wild type), Lys, Cys, Met, and Tyr enzymes, respectively. These results indicate that the amino acid substitution at position 112 affects the oxidation-reduction potential of the heme iron in cytochrome P450 cam , thereby diminishing the rate of electron transfer between the two metal centers. The rate of electron transfer from putidaredoxin to oxyferrous cytochrome P450 cam also diminished upon substitution of Arg 112 with a neutral amino acid.
Cytochrome P450 is a generic name given to a family of protoheme-proteins that metabolize a wide variety of natural and unnatural substances such as steroids, fatty acids, hydrocarbons, and xenobiotics. Among them, cytochrome P450 cam (P450 cam ) 1 of Pseudomonas putida (CYP101) has been the focus of intense mechanistic studies; its three-dimensional structure has been solved at 1.63-Å resolution by Poulos and co-workers (1,2), allowing us to make detailed analyses of the structure-function relationship. It catalyzes the hydroxylation of d-camphor to give 5-exo-hydroxycamphor as in the following scheme. The monooxygenation reaction requires, in addition to dcamphor and molecular oxygen, two reducing equivalents, which are transferred from NADH to P450 cam through a specific electron transfer system composed of NADH-putidaredoxin reductase (PdR), a flavoprotein, and putidaredoxin (Pd), an iron-sulfur (Fe 2 S 2 ) protein. In the reaction, Pd receives electrons from PdR and transfers them to P450 cam ; Pd serves as the direct electron donor for P450 cam .
When the mechanism for the electron transfer reaction between Pd and P450 cam was examined, reduced Pd and ferric P450 cam molecules were found to associate rapidly to form a bimolecular complex, followed by an intracomplex electron transfer giving ferrous P450 cam and oxidized Pd (3)(4)(5). Then, on the basis of results obtained from computer modeling of P450 cam -cytochrome b 5 complex, the binding site of Pd on P450 cam was suggested (6) to be in the proximal surface of P450 cam , which contains four basic amino acid residues (Arg 72 , Arg 112 , Lys 344 , and Arg 364 ). Stayton and Sligar (7) showed that neutralization of a positive charge at Arg 72 or Lys 344 by sitedirected mutagenesis resulted in a decrease in the binding affinity of P450 cam to Pd, although the changes in the values observed were rather small (1.5-fold at the maximum). More recently, however, Koga et al. (8) as well as Nakamura et al. (9) demonstrated that charge neutralization at Arg 112 , another basic amino acid among the four basic residues, evoked a dramatic decrease in the catalytic activity of P450 cam . Catalytic activities of Cys, Glu, and Gln mutants were less than 1% of that of the wild-type enzyme in a reconstituted assay system composed of NADH, PdR, Pd, and a mutant P450 cam . Thus, the presumed binding site in the proximal surface could be the real site for the binding of Pd to P450 cam at least in part.
In the present study, we further elucidate the role of Arg 112 in the electron transfer reaction from reduced Pd to ferric P450 cam by using site-directed mutants of P450 cam , in which the Arg residue was changed to a Lys, Cys, Tyr, or Met residue. Since they have different charges and hydrogen-bonding capacities with one another, we had hoped that their use might give us a clue to identify the role of the basic residue Arg 112 in the Pd-P450 cam interaction. The results revealed that the oxidation and reduction (redox) potential of the heme iron in P450 cam was greatly affected by the properties of an amino acid at 112position and that such changes in the redox potential were reflected in the rates of electron transfer between the two metal centers, i.e. the heme iron in P450 cam and nonheme iron in Pd. Possible mechanisms through which an amino acid substitution affects the redox potential are discussed. Finally, the cationic charge at Arg 112 was found to be important also in the second electron transfer, i.e. in the reduction of the ferrous oxygenated form of P450 cam (oxy-P450 cam ) by reduced Pd.

EXPERIMENTAL PROCEDURES
Enzyme Preparations and Catalytic Activity-The wild-type P450 cam and its mutants, prepared by the method of Kramer et al. (10), were expressed in Escherichia coli strain JM109 and purified with the procedures described previously (11). Purified preparations with an RZ value (A392/A280) greater than 1.45 were employed in this study. Pd and PdR were expressed also in E. coli strain JM109 and were purified to a homogeneous state according to the methods described by Gunsalus and Wagner (12). Monooxygenase activity of P450 cam was measured in a reconstituted assay system containing 1 mM d-camphor, 360 M NADH, 50 mM KCl, 14 M Pd, 0.12 M PdR, and an appropriate amount of P450 cam in 50 mM potassium phosphate buffer, pH 7.4, under normal atmospheric conditions at 20°C. The rate of reaction was determined by measuring both oxygen consumption and NADH oxidation rates simultaneously in a special cuvette described elsewhere (13). In most cases, the ratio of the two rates was 1:1, indicating that O 2 was consumed by a two-electron reduction process. When necessary, enzyme activity was determined by measuring the hydroxylated product of d-camphor (11). Only 5-exo-hydroxycamphor was detected as the hydroxylated product of d-camphor throughout this study.
Spectrophotometry and Stopped Flow Experiments-All spectrophotometric measurements were carried out in 50 mM potassium phosphate buffer, pH 7.4, containing 50 mM KCl and 1 mM d-camphor, hereafter called the standard buffer. Optical absorption spectra of the proteins were recorded with a Shimadzu spectrophotometer, model MPS-2000 (Kyoto, Japan). CD spectra of the enzymes were measured from 190 to 250 nm with a Jasco J-720 CD spectropolarimeter (Tokyo, Japan) at an ambient temperature. The buffer used for the CD measurements was 25 mM potassium phosphate buffer, pH 7.4, containing 25 mM KCl and 0.5 mM d-camphor.
The rate of reduction of ferric P450 cam by reduced Pd, i.e. the first electron transfer, was measured with a UNISOKU stopped flow spectrophotometer, model RSP-601 (Osaka, Japan). Usually, one reservoir of the stopped flow apparatus contained 2 M P450 cam in the standard buffer under anaerobic conditions, and the other contained 3-54 M reduced Pd in the standard buffer equilibrated with 1.0 atmosphere CO. The reaction was started by mixing the two solutions and followed by measuring the formation of the ferrous CO form of P450 cam at 446 nm (4). Changes in absorbance were digitized on a Yokogawa DL1200 oscilloscope (Tokyo, Japan). Reduced Pd was prepared by the addition of 360 M NADH and a catalytic amount of PdR (about 0.1 M) to the solution of oxidized Pd under anaerobic conditions. An oxygen-scavenging system composed of glucose (60 mM), glucose oxidase (0.1 mg/ml; Sigma), and catalase (3,000 units/ml; Sigma) was included in the medium to ensure the anaerobiosis (4, 5). All measurements were done at 20°C.
Reduction of oxy-P450 cam by reduced Pd, i.e. the second electron transfer, was assessed by following the spectral changes of oxy-P450 cam (380 -540 nm) after mixing the oxy form (5 M) with reduced Pd (18 M) in the stopped flow/rapid scan apparatus at 4°C. Oxy-P450 cam was prepared by mixing the ferrous P450 cam solution with an equal amount of an O 2 -saturated buffer at 4°C and was immediately transferred to a reservoir of the stopped flow apparatus. The ferrous P450 cam was prepared by adding a minimum volume of 20 mM sodium dithionite solution to ferric P450 cam under anaerobic conditions. The reduced Pd was prepared as described above. The buffer system employed in these experiments was the same as that used in the measurements of the first electron transfer rate.
Kinetic Modeling of the First Electron Transfer-The reduction of ferric P450 cam by reduced Pd has been shown to be represented by a two-step model presented in Equation 1 (4,14).
For the mechanism in Equation 1, time-dependent changes in the concentration of the reactants (A and B), the reaction intermediate (C), and the product (D) are defined as in Equations 2 and 3.
In the present experiment, the associating species A and B correspond to ferric P450 cam and reduced Pd, respectively, and the species C is an equimolar complex of ferric P450 cam and reduced Pd. Then D represents the product of the reaction, i.e. oxidized Pd-ferrous P450 cam complex. The complex further dissociates into oxidized Pd and ferrous P450 cam , but this reaction is not considered here; the ferrous P450 cam formed is immediately trapped as its CO adduct in the presence of an excess amount of CO either in the complexed or dissociated state (3,4). Thus, the formation of the product (D) could be monitored by measuring the amount of the ferrous CO complex of P450 cam at 446 nm. Under such conditions, the dissociation of the CO-ferrous adduct is very slow, and hence we can assume k Ϫ2 ϭ 0 for a kinetic modeling.
The differential equations of Equations 2 and 3 were iteratively solved with a digital computer by using the fourth-order Runge-Kutta numerical method at 2-s intervals. The initial concentrations of P450 cam and Pd were determined by measuring absorption spectra of both proteins, and parameters k 1 , k Ϫ1 , and k Ϫ2 were set variable to obtain the best fit to the experimental data. The dissociation constant of reduced Pd-ferric P450 cam complex can be obtained by dividing k Ϫ1 by k 1 (K d ϭ k Ϫ1 /k 1 ).
Oxidation and Reduction Potentials-The redox potential of P450 cam was measured under anaerobic conditions by the method of Makino et al. (15) with minor modifications. In brief, the reaction mixture for the measurements contained about 46 M of the wild-type or a mutant P450 cam and the following ingredients in 50 mM potassium phosphate buffer, pH 7.4: 50 mM KCl, 500 M d-camphor, proflavin sulfate, EDTA, ␣-hydroxyphenazine, pyocyanine, phenosafranine, and safranine T. Ferric P450 cam was reduced by illuminating the sample with white tungsten light (150 W) under anaerobic conditions at 20°C. A microcombination electrode from Ingold (Pt-4800-M5), which was calibrated by measuring the potential of phenosafranine, was used to monitor the potential of the Fe 3ϩ /Fe 2ϩ couple in P450 cam .

RESULTS
Spectra and Activities of P450 cam Mutants- Fig. 1 shows absorption spectra of ferric, ferrous, and ferrous CO forms of The spectra were almost indistinguishable from those of the wild-type enzyme (11,12) and also from those of other 112position mutants such as Lys, Cys, and Tyr enzymes; the peak positions of the enzymes agreed well within plus or minus 0.5 nm, and their extinction coefficients were not greatly different from one another. CD spectra (190 -250 nm) of the mutant enzymes were also indistinguishable from that of the wild-type enzyme (data not shown). These results indicate that the mutation at position 112 had little influence on the structure of heme and its vicinity of P450 cam as well as on the polypeptide backbone folding. The finding was in conformity with the results of a computer simulation study on the structure of the Cys mutant described by Koga et al. (8), where little conformational change was expected as compared with that of the wild-type enzyme.
The catalytic activity of the enzyme was dramatically affected by these mutations. As shown in Table I, the rates of oxygen consumption by Cys, Met, and Tyr mutants were 1 ⁄1,000, 1 ⁄764, and 1 ⁄1,625 of that by the wild-type (Arg) enzyme, respectively. Meanwhile, the replacement of Arg by Lys, which has a cationic charge, did not result in such a big decrease in the oxygen consumption rate; it diminished to 1/6.5 (200 min Ϫ1 ) of the wild-type enzyme (1,300 min Ϫ1 ), indicating that a positive charge rather than the other properties of the amino acid residue at 112-position is necessary to maintain the high catalytic activity.
Table I also showed that, although the rate of oxygen consumption was reduced substantially by the charge neutralization, most of the oxygen consumed by these mutants was utilized to hydroxylate d-camphor. As depicted in Table I, the ratios of hydroxylated camphor to consumed oxygen were kept rather high (50 -88%). On the other hand, the ratios of hydrogen peroxide formed to oxygen consumed were small (12-30%). Thus, the mutation at this position did not cause much uncoupling of NADH oxidation from monooxygenation as compared with that observed for the mutants at 252-position (threonine) (11,16). The increase in the uncoupling ratio to 30% for Tyr enzyme may appear large, but it is not as much as the decrease in the rate of oxygen consumption; the oxygen consumption rate dropped to less than 0.01% of that by wild-type enzyme.
Effects of Mutation on the First Electron Transfer- Figs. 2A  and B show time courses of the reaction, in which 1 M ferric Lys mutant of P450 cam was reduced to a ferrous state by either 1.5 or 27 M of reduced Pd. The open circles represent experimental data points, and the lines through the points are sim-ulated curves obtained by assuming the mechanism in Equation 1. The details for the simulation will be discussed later, but both experimental and simulated curves fitted well to a singleexponential curve in accordance with that for the wild-type P450 cam (4). Here we show only the time courses for the Lys mutant, but the reaction of all other P450 cam with reduced Pd proceeded in a similar way; they apparently followed first-order kinetics. Then we plotted apparent first-order rate constants (k obs ) as a function of reduced Pd concentration (Fig. 3). As seen, all mutant enzymes exhibited significantly smaller k obs values than that of the wild-type enzyme, and changes in k obs reached to a plateau at high concentrations of Pd. Such a nonlinear behavior is consistent with a two-step mechanism in Equation 1 consisting of a rapid diprotein complex formation (second-order reaction) and following intracomplex electron transfer (first-order reaction), where the latter becomes the rate-limiting step at higher concentrations of Pd.
To determine the rate constants, k 1 , k Ϫ1 , and k 2 in Equation 1, we performed two-step model fitting onto the time course data obtained for each P450 cam species. The best-fit values thus obtained for the rate constants in the reaction of each P450 cam species at 20°C were summarized in Table II, and the time course curves were simulated using such values for the Lys mutant as have been illustrated already in Fig. 2, (A and B) as examples. The results revealed that both the dissociation constant K d and the electron transfer rate k 2 for Cys, Met, and Tyr mutants were 500-2500-fold and 1 ⁄10-1 ⁄250 that of the wild-type enzyme, respectively. Thus, the charge neutralization at position 112 caused significant decreases in the affinity of reduced Pd for ferric P450 cam together with the decreases in the rate of intracomplex electron transfer. On the other hand, K d and k 2 of the Lys mutant were not greatly different from those of the wild-type enzyme, indicating that the presence of a positive  Effects of Mutation on the Redox Potential-Ferric forms of all P450 cam species employed in this study showed a typical behavior of a one-electron carrier during the reductive titration in the presence of 1 mM d-camphor. Midpoint potentials (E m in mV) of five P450 cam species thus obtained are also listed in Table I. The value of E m ϭ Ϫ138 mV for the wild-type P450 cam was lowered to Ϫ162, Ϫ182, Ϫ200, and Ϫ195 mV upon substitution of Lys, Cys, Met, and Tyr for Arg, respectively. It should be noted that the values for the latter three mutants are close to the value of Ϫ215 mV, the midpoint potential of their electron donor, Pd. Then the decrease in E m (24 -62 mV) appeared not correlative with a single property of amino acid such as volume, length, and charge of the side chain, but with their combinations.
Redox Potential and Electron Transfer Rate (k 2 )- Fig. 4 shows a plot of intracomplex electron transfer (k 2 ) against the changes in the redox potential of P450 cam . In addition to the data obtained in the present study (closed circle), those from a study by Fisher and Sligar (18) who examined changes in the redox potential of the wild-type enzyme upon combination with various substrate analogs (open circle) were employed in this plot. A trend is clearly seen; the decrease in the redox potential accompanies the decrease in the electron transfer rate, which is expressed in a logarithmic scale. On the basis of these observations and theoretical considerations previously reported (19), we have concluded that the decrease in the rate of intracomplex electron transfer observed in this study is mainly due to the lowered redox potential of the heme iron.
Effects of Mutation on the Second Electron Transfer-The reduction of oxy-P450 cam , a ternary complex of oxygen and d-camphor with the ferrous enzyme, by reduced Pd is known to yield the monooxygenated reaction product, 5-exo-hydroxycamphor bound to ferric P450 cam . Subsequent release of the hydroxycamphor from the enzyme was followed by the binding of another d-camphor molecule to regenerate the d-camphorbound ferric P450 cam . The rate of second electron transfer was therefore assessed by monitoring the formation of ferric P450 cam after mixing oxy-P450 cam with reduced Pd. Fig. 5A shows changes in absorbance at 404 nm due to conversion of the oxy form of the wild-type P450 cam to the ferric form as a function of time. Since 404 nm is an isosbestic point of substrate-free and -bound forms of ferric enzyme, changes in absorbance at this wavelength can represent changes in both substrate-free and -bound ferric enzymes, although practically no substrate-free form is present under the experimental conditions, i.e. in the presence of 1 mM d-camphor. Then analyses revealed that the traces fitted to a single-exponential kinetics with a half-time t1 ⁄2 ϭ 9.4 ms. The reaction between reduced Pd

TABLE II
Best fit parameters for the electron transfer reaction from reduced putidaredoxin to various ferric cytochromes P450 cam The parameters were obtained by fitting Equations 2 and 3 (see "Experimental Procedures") to the observed kinetic traces as described in the text. The values of the dissociation constant were calculated according to the equation, K d ϭ k Ϫ1 /k 1 . a -, a wide range of k 1 and k Ϫ1 showed identical fits within experimental error, although calculated K d from those k 1 and k Ϫ1 was found to be constant. Thus only K d was listed for these mutants.
b The reaction employing Pd concentration below 2 M, which is the concentration after mixing on the stopped flow apparatus, was slow to observe whole kinetic traces by the data acquisition system of our instruments. Thus, the kinetic traces at Pd concentrations higher than 2 M were employed for the analysis.  Tables I and II. The values k 0 and k correspond to k 2 for the wild-type and mutant P450 cam , respectively. ⌬E is the difference in redox potential between the mutant and the wildtype P450 cam ; ⌬E ϭ E of the mutant P450 cam Ϫ E of the wild-type P450 cam . The data represented by open circles are taken from Fisher and Sligar (18). In the latter case, k and k 0 correspond to the reduction rate of P450 cam bound to substrate analogs and d-camphor, respectively. ⌬E was the difference in redox potential between P450 cam associated with substrate analogs and d-camphor. Differences in redox potentials (⌬E) and ratios (k/k 0 ) of the electron transfer rate are plotted. and the oxy form of the Lys enzyme showed a similar but slower decay of the oxy form (t1 ⁄2 ϭ 130 ms) as compared with that of the wild-type enzyme.
A dramatic effect on the kinetics was found when Arg 112 was replaced with Cys, Met, or Tyr. The trace with the Tyr mutant was shown in Fig. 5B, where changes in absorption at 404 nm were not detected even at 400 ms after the mixing. The situation was the same for the Cys and Met enzymes. These results clearly indicated that the removal of a positive charge at position 112 reduced the rate of electron transfer from reduced Pd to oxy-P450 cam . DISCUSSION We found in this study that charge neutralization at Arg 112 , a surface amino acid residue of P450 cam , affected the redox potential of the heme iron, which is buried in the interior of the protein; the redox potential of the wild-type P450 cam , Ϫ138 mV, was lowered to Ϫ182 to Ϫ200 mV in the mutant enzymes having a neutral amino acid residue such as Cys, Met, or Tyr at the 112-position. It should be noted that the redox potential of Pd, the electron donor for P450 cam , is Ϫ215 mV under comparable conditions (15). The mutation also affected the rate of electron transfer from reduced Pd to either ferric or oxyferrous P450 cam and hence overall catalytic activity. It has been shown that the electron transfer process, especially that to oxy-P450 cam , is the rate-limiting step in the overall catalytic reaction catalyzed by P450 cam . The effect of amino acid substitution on both electron transfer and catalytic rates can be regarded as the secondary to the effect on the redox potential, since redox potential has been known to be an important determinant of the rate of outer sphere electron transfer between two redox centers (19,20). A question thus arises as to the mechanisms by which mutation causes the changes in the redox potential.
First of all, an electrostatic interaction of the redox centers with charged groups of the protein must be considered (21). For example, Caffrey and Cusanovich (22) replaced some surface Lys residues of cytochrome c 2 with negatively charged Asp or Glu and showed that the redox potentials (Fe 3ϩ /Fe 2ϩ couple) of the mutants decreased by 11-14 mV. The decrease in redox potential can be due to an increase in the stability of the ferric state as a result of interaction between the positively charged ferric heme iron and a more negative (less positive) electrostatic field of the protein. The second possibility is the changes in hydrogen-bonding interactions of the heme propionate with amino acid residues in the apoprotein moiety. In their crystallographic studies on P450 cam , Poulos et al. (1) have shown that the guanidino group of Arg 112 is hydrogen-bonded to the oxygen atom of 6-propionate of the iron protoporphyrin. Thus, the substitution of Arg 112 with Lys, Tyr, Met, and Cys leads to the modification or elimination of the hydrogen-bonding interactions between the heme propionate and an amino acid residue at position 112. The interactions through the hydrogen bond will withdraw a negative charge from the propionate group, thereby destabilizing the positive charge on the heme iron. Hence, a weaker hydrogen bond or elimination of the hydrogen bond in the mutant enzymes reduces the degree of charge withdrawal from the propionate and results in the stabilization of the Fe 3ϩ heme iron. Such an idea was supported by a recent study of Davies et al. (23) on cytochrome c. They stated that the loss of a single hydrogen bond resulted in a ϳ20 mV drop in the redox potential.
Third, effects on the thiolate axial ligand must also be taken into consideration. Above mentioned propionate is also hydrogen-bonded to the imidazole nitrogen of His 355 , which is the second from the thiolate axial ligand, Cys 357 . Lys, Cys, Met, or Tyr incorporated to the 112-position in place of Arg possibly alters the electrostatic and/or conformational states of the charged carboxyl group of the heme propionate. Such changes occurring at the propionate side chain may induce more or less perturbation of the thiolate ligand through a hydrogen bond between the propionate and His 355 and/or through peptide bonds linking this His and the axial ligand.
In addition to the changes in redox potential, we have also shown that the charge neutralization at the 112-position of P450 cam reduces the affinity of ferric P450 cam to Pd (Table II). Electrostatic forces have long been considered to contribute to the interaction between Pd and P450 cam ; positive charges on the surface of P450 cam have been considered to interact with corresponding negative charges on Pd (4,5,6,24). On the other hand, charge neutralization at the 72-and 344-positions resulted in only a slight increase in the affinity (ϳ1.5-fold at the maximum) (7). Furthermore, we recently found that substitution of Arg 364 with Met perturbed the enzyme activity only sluggishly. 2 Thus, the role of Arg 112 in the P450 cam -Pd interaction is unique among the surface amino acid residues so far examined. The reason for this uniqueness of Arg 112 is unknown at present, but it is possible that only Arg 112 can form a specific ion pair with an anionic surface residue of Pd, while the others do not. Since a number of negatively charged groups are located on the surface of the protein, including Asp 9 , Asp 34 , Asp 38 , Glu 77 , Asp 100 , and Asp 103 (25,26), we suggest that one of these residues interact(s) electrostatically with Arg 112 of P450 cam . We have also demonstrated that the second electron transfer was greatly affected by the mutation at the 112-position of P450 cam . In this connection, the following difference has been noted between the reduction of ferric P450 cam and that of oxy-P450 cam , i.e. the first and the second reduction of P450 cam in the reaction cycle. Certain reductants such as spinach ferredoxin, adrenodoxin, and dithionite, which have lower redox potentials than Pd, can reduce ferric P450 cam but do not reduce oxy-P450 cam to give the hydroxylated product, d-camphor (27). Later, further studies done by Peterson and co-workers (4,5) revealed that the effect of KCl upon the rates of electron transfer (k obs ) was different between the two processes; in the first electron transfer, KCl concentrations had no effect on k obs until it reached about 0.5 M, above which k obs declined with the increase in KCl concentration. For the second electron transfer, on the other hand, there was a large decrease in k obs before KCl concentration reached to 0.5 M. Studies on the active site mutants of P450 cam by us (16,28) and by Gerber and Sligar (29,30) have also shown a dissimilarity between the two processes. In these studies, it has been demonstrated that the mutation of Asp 251 to Gly, Ala, or Asn does not reduce the rate of the first process, but dramatically slows down the second reduction rate. While proton transfer also needs to be considered in the latter process (28,30,31), the observation with the Asp 251 mutants seems to suggest that the Pd binding site and/or electron transfer pathway for the second electron transfer is different from those for the first process. However, our results in Table II and Fig. 5 have clearly shown that the replacement of Arg 112 with a neutral amino acid (i.e. Met, Cys, or Tyr) inhibits not only the first electron transfer but also the second process. This implies that Arg 112 also contributes to the reduction of oxyferrous P450 cam . Further experiments are obviously necessary to solve these and other problems in the mechanism(s) of electron transfer reaction from Pd to P450 cam .