Dynamics underlying hydroxylation selectivity of cytochrome P450cam

Structural heterogeneity and the dynamics of the complexes of enzymes with substrates can determine the selectivity of catalysis; however, fully characterizing how remains challenging as heterogeneity and dynamics can vary at the spatial level of an amino acid residue and involve rapid timescales. We demonstrate the nascent approach of site-specific two-dimensional infrared (IR) spectroscopy to investigate the archetypical cytochrome P450, P450cam, to better delineate the mechanism of the lower regioselectivity of hydroxylation of the substrate norcamphor in comparison to the native substrate camphor. Specific locations are targeted throughout the enzyme by selectively introducing cyano groups that have frequencies in a spectrally isolated region of the protein IR spectrum as local vibrational probes. Linear and two-dimensional IR spectroscopy were applied to measure the heterogeneity and dynamics at each probe and investigate how they differentiate camphor and norcamphor recognition. The IR data indicate that the norcamphor complex does not fully induce a large-scale conformational change to a closed state of the enzyme adopted in the camphor complex. Additionally, a probe directed at the bound substrate experiences rapidly interconverting states in the norcamphor complex that explain the hydroxylation product distribution. Altogether, the study reveals large- and small-scale structural heterogeneity and dynamics that could contribute to selectivity of a cytochrome P450 and illustrates the approach of site-selective IR spectroscopy to elucidate protein dynamics.     

Theoretical study of the product specificity in the hydroxylation of camphor, norcamphor, 5,5-difluorocamphor, and pericyclocamphanone by cytochrome P-450cam

The hydroxylations of d-camphor, norcamphor, pericyclocamphanone, and 5,5-difluorocamphor by cytochrome P-450cam have been examined using theoretical methods to identify and characterize properties which determine product specificity. Experimental results indicate that each molecule is hydroxylated with quite different regio-specificity when metabolized by P-450cam. This result is surprising in view of their overall structural similiarity. Herein we report the results of calculations on d-camphor and three of its analogues which suggest that all of these molecules should, when metabolized by P-450cam, form hydroxylation products and predict the product distribution for each. Our conclusions are based on two fundamental criteria which are consistent with a generally accepted radical mechanism in determining product specificity in these molecules: 1) relative heats of formation of the radicals formed by abstracting a hydrogen, and 2) orientation of the substrate molecule with respect to the putative active oxygen species bound to iron. Our results explain the experimental observations for camphor and 5,5-difluorocamphor but disagree with original published results for norcamphor and pericyclocamphanone. In light of our results, new experiments have been performed for norcamphor and the original data reexamined for pericyclocamphanone. Our predictions have recently been experimentally confirmed for norcamphor, and unpublished data (Dr. S. Sligar) suggest that the same is true for pericyclocamphanone.     

Structural alterations of the heme environment of cytochrome P450cam and the Y96F mutant as deduced by resonance Raman spectroscopy

Resonance Raman spectroscopy at 2.5cm(-1) resolution was used to probe differences in wild-type and Y96F mutant P450cam (CYP101), both with and without bound camphor or styrene substrates. In the substrate-free state, the spin state equilibrium is shifted from 6-coordinate low spin (6CLS) toward more 5-coordinate high spin (5CHS) when tyrosine-96 in the substrate pocket is replaced by phenylalanine. About 25% of substrate-free Y96F mutant is 5CHS as opposed to 8% for substrate-free wild-type P450cam. Spin equilibrium constants calculated from Raman intensities indicate that the driving force for electron transfer from putidaredoxin, the natural redox partner of P450cam, is significantly smaller on styrene binding than for camphor binding. Spectral differences suggest that there is a tilt in camphor toward the pyrrole III ring on Y96F mutation. This finding is consistent with the altered product distribution found for camphor hydroxylation by the Y96F mutant relative to the single enantiomer produced by the wild-type enzyme.     

Autooxidation and hydroxylation reactions of oxygenated cytochrome P-450cam.

Oxy-ferrous substrate-bound cytochrome P-450cam (mrsO2) autooxidizes in the absence of its specific effector protein, putidaredoxin, without hydroxylating the substrate, camphor. The autooxidation is first order with an activation energy of 17 kcal mol-1 at 25 degrees, pH 7.0. Substrate removal and low pH accelerate the reaction. The product, 5-exo-OH camphor, and a nonhydroxylated pseudosubstrate, norcamphor, stabilize the complex in a manner similar to camphor. Increased oxidation rate of mrsO2 and substrate hydroxylation are induced by putidaredoxin, rebredoxin, cytochrome b5, and the apoproteins of the latter two. Dihydrolipoic acid and other dithiols also replace putidaredoxin as effector molecules, but 1000-fold higher concentrations are required. Effector molecules do not increase the autooxidation rate of mrsO2 unless camphor, norcamphor, or another pseudosubstrate is present. Kinetic evidence is presented showing that an active complex between mrsO2 and effector is a required intermediate in mixed function oxidation.     

Substrate mobility in a deeply buried active site: analysis of norcamphor bound to cytochrome P-450cam as determined by a 201-psec molecular dynamics simulation.

While cytochrome P-450cam catalyzes the hydroxylation of camphor to 5-exo-hydroxycamphor with 100% stereospecificity, norcamphor is hydroxylated by this enzyme yielding 45% 5-exo-, 47% 6-exo-, and 8% 3-exo-hydroxynorcamphor (Atkins, W.M., Sligar, S.G., J. Am. Chem. Soc. 109:3754-3760, 1987). The present study describes a 201-psec molecular dynamics (MD) stimulation of norcamphorbound cytochrome P-450cam to elucidate the relationship between substrate conformational mobility and formation of alternative products. First, these data suggest that the product specificity is, at least partially, due to the mobility of the substrate within the active site. Second, the high mobility of norcamphor in the active site leads to an average increase in separation between the heme iron and the substrate of about 1.0 A; this increase in separation may be the cause of the uncoupling of electron transfer when norcamphor is the substrate. Third, the active site water located in the norcamphorbound crystal structure possesses mobility that correlates well with the spin-state equilibrium of this enzyme-substrate complex.     

A recombinant Escherichia coli whole cell biocatalyst harboring a cytochrome P450cam monooxygenase system coupled with enzymatic cofactor regeneration

A cytochrome P450cam monooxygenase (P450cam) system from the soil bacterium Pseudomonas putida requires electron transfer among three different proteins and a cofactor, nicotinamide adenine dinucleotide (NADH), for oxygenation of its natural substrate, camphor. Herein, we report a facile way to significantly enhance the catalytic efficiency of the P450cam system by the coupling of its native electron transfer system with enzymatic NADH regeneration catalyzed by glycerol dehydrogenase (GLD) in Escherichia coli whole cell biocatalysts. Recombinant E. coli harboring the P450cam system, but lacking GLD, exhibited little activity for camphor hydroxylation. In contrast, coexpression of GLD with the proteinaceous electron transfer components of P450cam resulted in about tenfold improvement in the substrate conversion, implying that the whole cell biocatalyst utilized molecular oxygen, endogenous NADH, and glycerol in the cell for catalysis. The addition of glycerol to the reaction media further promoted camphor hydroxylation, suggesting that exogenous glycerol is also available for GLD in the host cell and actively participates in the catalytic cycle. These results clearly show the utility of GLD towards functional reconstruction of the native P450cam system. The present approach may also be useful for E. coli whole cell biocatalysts with the other NADH-dependent oxygenases and oxidoreductases.     

Substrate modulates compound I formation in peroxide shunt pathway of Pseudomonas putida cytochrome P450(cam)

The active oxygenating intermediate, a ferryl-oxo-(II) porphyrin cation radical (compound I), in substrate-bound cytochrome P450(cam) (P450(cam)) has eluded detection and kinetic analysis for several decades. Upon rapid mixing of peroxides-H(2)O(2) and m-CPBA with substrate-bound forms of P450(cam), we observed an intermediate with spectral features characteristic of compound I. Unlike in H(2)O(2), kinetic investigation on the reaction of m-CPBA with various substrate (camphor, adamantone, and norcamphor)-bound P450(cam) and its Y96A mutant shows a preferential binding of the aromatic end group of m-CPBA to the active-site of the enzyme and modulation of compound I formation by the local environment of heme active-site. The results presented in this paper describe the importance of heme environment in modulating formation of compound I, and form the first kinetic analysis of this intermediate in the peroxide shunt pathway of substrate-bound P450(cam).     

Regiochemistry of Camphor Analog Oxidation by Pseudomonas putida

Pseudomonas putida cooxidized norcamphor and pericyclocamphanone to hydroxylated and lactonized products during growth on camphor. Norcamphor was hydroxylated at the 5 position, similar to the corresponding process in camphor, but pericyclocamphanone was oxidized at the 6 position. We conclude that the regiochemistry of the hydroxylation may be substrate controlled.     

Active site mutations of cytochrome p450cam alter the binding, coupling, and oxidation of the foreign substrates (R)- and (s)-2-ethylhexanol

Three factors are of primary importance with respect to designing efficient P450 biocatalysts. (1) The substrate must be oxidized at a significant rate. (2) The regioselectivity must heavily favor the desired product. (3) The enzyme must use the majority of the reducing equivalents from NADH or NADPH to produce product. The reaction we chose to study was oxidation of 2-ethylhexanol to 2-ethylhexanoic acid by P450cam. We examined four active site mutations: F87W, Y96W, T185F, and L244A. The mutations were chosen to improve 2-ethyhexanoic acid production by decreasing active site volume, increasing active site hydrophobicity, and improving stereoselectivity. The F87W and Y96W mutations improved regioselectivity, giving almost exclusively the desired product. The T185F mutation improved coupling of NADH to product formation. The L244A mutation altered the stereoselectivity of 2-ethylhexanoic acid production. These results indicate that active site mutations of P450cam can alter catalysis of 2-ethylhexanol.     

The structural basis for substrate-induced changes in redox potential and spin equilibrium in cytochrome P-450CAM

The crystal structures of cytochrome P-450CAM complexed with the alternative substrates norcamphor and adamantanone have been refined at 2.0-A resolution and compared with the native, camphor-bound form of the enzyme. Norcamphor lacks the 8-, 9-, and 10-methyl groups of camphor. Thus, specific interactions between these groups and phenylalanine 87 and valines 247 and 295 are missing in the norcamphor complex. As a result, norcamphor binds about 0.9 A further from the oxygen-binding site than does camphor, which allows sufficient room for a water molecule or hydroxide ion to remain coordinated with the heme iron atom. The larger adamantanone occupies a position closer to that of camphor and, as in the camphor-bound enzyme, the heme iron remains pentacoordinate with no solvent molecule coordinated as a sixth ligand. A comparison of crystallographic temperature factors indicates that norcamphor is more "loosely" bound than are either camphor or adamantanone, as might be expected from the relative sizes of the different substrates. The looser fit of norcamphor in the active-site pocket results in a less specific pattern of hydroxylation. The presence of an aqua ligand is the likely structural basis for the norcamphor-P-450CAM complex having both a lower redox potential and higher percentage of low-spin heme than do either the camphor-P-450CAM or adamantanone-P-450CAM complexes.     

Protein engineering of cytochrome p450(cam) (CYP101) for the oxidation of polycyclic aromatic hydrocarbons

Mutations of the active site residues F87 and Y96 greatly enhanced the activity of cytochrome P450(cam) (CYP101) from Pseudomonas putida for the oxidation of the polycyclic aromatic hydrocarbons phenanthrene, fluoranthene, pyrene and benzo[a]pyrene. Wild-type P450(cam) had low (<0.01 min(-1)) activity with these substrates. Phenanthrene was oxidized to 1-, 2-, 3- and 4-phenanthrol, while fluoranthene gave mainly 3-fluoranthol. Pyrene was oxidized to 1-pyrenol and then to 1,6- and 1,8-pyrenequinone, with small amounts of 2-pyrenol also formed with the Y96A mutant. Benzo[a]pyrene gave 3-hydroxybenzo[a]pyrene as the major product. The NADH oxidation rate of the mutants with phenanthrene was as high as 374 min(-1), which was 31% of the camphor oxidation rate by wild-type P450(cam), and with fluoranthene the fastest rate was 144 min(-1). The oxidation of phenanthrene and fluoranthene were highly uncoupled, with highest couplings of 1.3 and 3.1%, respectively. The highest coupling efficiency for pyrene oxidation was a reasonable 23%, but the NADH turnover rate was slow. The product distributions varied significantly between mutants, suggesting that substrate binding orientations can be manipulated by protein engineering, and that genetic variants of P450(cam) may be useful for studying the oxidation of polycyclic aromatic hydrocarbons by P450 enzymes.     

Spring-loading the active site of cytochrome P450cam

A hydrogen bond network has been identified that adjusts protein-substrate contacts in cytochrome P450(cam) (CYP101A1). Replacing the native substrate camphor with adamantanone or norcamphor causes perturbations in NMR-detected NH correlations assigned to the network, which includes portions of a β sheet and an adjacent helix that is remote from the active site. A mutation in this helix reduces enzyme efficiency and perturbs the extent of substrate-induced spin state changes at the haem iron that accompany substrate binding. In turn, the magnitude of the spin state changes induced by alternate substrate binding parallel the NMR-detected perturbations observed near the haem in the enzyme active site.     

Mutations of glutamate-84 at the putative potassium-binding site affect camphor binding and oxidation by cytochrome p450cam.

Cytochrome P450cam (CYP101) from Pseudomonas putida is unusual among P450 enzymes in that it exhibits co-operative binding between the substrate camphor and a potassium ion. This behaviour has been investigated by mutagenesis of Glu84, a surface residue which forms part of the cation-binding site. Substitutions that neutralize or reverse the charge of this side chain are shown to disrupt the co-operativity of potassium and camphor binding by P450cam, and also to influence the catalytic activity. In particular, replacement of Glu84 by positively charged residues such as lysine results in increased high-spin haem fractions and camphor turnover activities in the absence of potassium, along with decreased camphor dissociation constants. However, in the presence of potassium the camphor dissociation constants of these mutants are significantly increased compared with the wild-type, although the camphor turnover activities remain marginally higher. In contrast, substitution by aspartate results in tighter binding of both potassium and camphor, but has little effect on the enzymatic activity. In all cases the reaction remains essentially 100% coupled and gives 5-exo-hydroxycamphor as the only product. These results suggest that an anionic side chain at the 84 position is crucial for the co-operativity of camphor and cation binding, and that the physiological role for potassium binding by cytochrome P450cam is to promote camphor binding even at the expense of turnover rate, thus allowing the organism to utilize low environmental concentrations of this substrate for growth.     

Molecular Dynamics Simulations Indicate that F87W,T185F-Cytochrome P450cam May Reductively Dehalogenate 1,1,1-Trichloroethane

Abstract

Cytochrome P450cam is capable of reductively dehalogenating several chlorinated alkanes at low, but measurable, rates. In previous investigations of structure-function relationships in this enzyme using molecular dynamics simulations, we noticed that 1,1,1-trichloroethane (TCA) exhibits a very high degree of mobility in the active site due to its smaller molecular volume relative to the native substrate, camphor(1,2). Several amino acid sidechains lining the active site also exhibit significant dynamic fluctuations, possibly as a result of poor steric complementarity to TCA. Guided by these results, we modeled double (F87W, T185F) and triple (F87W, T185F, V295I) mutants of P450cam, which provide additional bulk in the active site and increase the frequency of heme-substrate collision. Molecular dynamics simulations (300 ps on each protein) indicate that these mutants do not significantly perturb the three-dimensional fold of the enzyme, or local structure in the region of the active site. Both mutants bind the substrate more stably near the heme than the wild-type. Interestingly, however, the bulkier triple mutant seems to actually inhibit heme-substrate interactions relative to the double mutant. Over the final 200 ps of simulation, TCA is within 1 Å of nonbonded contact with the heme 25% more often in the double mutant versus the wild-type. The triple mutant, on the other hand, binds TCA within 1 Å of the heme only 15% as often as the wild-type. These results indicate that the double mutant may reductively dehalogenate TCA, a property not observed for the native protein. Implications for other experimentally measurable parameters are discussed.     

Regioselectivity in the cytochromes P-450: control by protein constraints and by chemical reactivities

Three alicyclic compounds (D-camphor, adamantanone, adamantane) were found to be hydroxylated by the cytochrome P-450 isoenzymes P-450cam and P-450LM2. With P-450cam as the catalyst only one product was formed from each of the substrates: 5-exohydroxycamphor, 5-hydroxyadamantanone, and 1-adamantanol. With P-450LM2 as the catalyst, two or more isomeric products were formed from each substrate: 3-endo-, 5-exo-, and 5-endo-hydroxycamphor; 4-anti- and 5-hydroxyadamantanone; and 1- and 2- adamantanol. The products from P-450cam hydroxylations were found to be isosteric with one another, suggesting that each of them was attacked at a topologically congruent position within a rigid enzyme-substrate complex. The distribution of products from P-450LM2 hydroxylations, on the other hand, were similar to the distributions expected during solution-phase hydroxylations. Thus, it would appear that the complex which P-450LM2 forms with its substrate allows considerable movement of the substrate molecule, such that most of the hydrogens in the substrate are exposed to the enzymatic hydrogen abstractor. Under these conditions, the distribution of products more nearly reflects the rank order of chemical reactivities of the various hydroxylatable positions, with only a moderate protein-based steric constraint being expressed. These suggestions were also evident in the tightness of binding of the substrates to the two enzymes and in the magnitude of coupling between the substrate binding and the spin-state equilibria. Thus, the product from P-450cam-catalyzed hydroxylation may be predicted by a consideration of the relation of the topology of the prospective substrate to that of D-camphor. The products from P-450LM2-catalyzed hydroxylations, on the other hand, may be approximately predicted from the chemical reactivities of the various abstractable hydrogens in the prospective substrate.     

Crystal structure of substrate-free Pseudomonas putida cytochrome P-450

The crystal structure of Pseudomonas putida cytochrome P-450cam in the substrate-free form has been refined at 2.20-A resolution and compared to the substrate-bound form of the enzyme. In the absence of the substrate camphor, the P-450cam heme iron atom is hexacoordinate with the sulfur atom of Cys-357 providing one axial heme ligand and a water molecule or hydroxide ion providing the other axial ligand. A network of hydrogen-bonded solvent molecules occupies the substrate pocket in addition to the iron-linked aqua ligand. When a camphor molecule binds, the active site waters including the aqua ligand are displaced, resulting in a pentacoordinate high-spin heme iron atom. Analysis of the Fno camphor - F camphor difference Fourier and a quantitative comparison of the two refined structures reveal that no detectable conformational change results from camphor binding other than a small repositioning of a phenylalanine side chain that contacts the camphor molecule. However, large decreases in the mean temperature factors of three separate segments of the protein centered on Tyr-96, Thr-185, and Asp-251 result from camphor binding. This indicates that camphor binding decreases the flexibility in these three regions of the P-450cam molecule without altering the mean position of the atoms involved.     

Differential behavior of the sub-sites of cytochrome 450 active site in binding of substrates, and products (implications for coupling/uncoupling)

The cytochrome P450 catalyzes hydroxylation of many substrates in the presence of O(2) and specific electron transport system. The ternary complex S-Fe(+)O(2) with substrate and O(2) bound to their respective sites on the reduced enzyme is an important intermediate in the formation of the hydroxylating species. Then the active site may be considered as having two sub-sites geared for entirely different types of functionally relevant interactions. The two sites are the substrate binding site, the specific protein residues (Site I), and the L(6) position of the iron (Site II) to which O(2) binds upon reduction. In the ferric enzyme, when substrate binds to Site I, the low spin six-coordinated P450 is converted to the readily reducible high spin five coordinated state. Certain amines and OH compounds, such as products of P450-catalyzed reactions, can bind to Site II resulting in six coordinated inhibited complexes. Then the substrate and product interactions with the two sub-sites can regulate the functional state of the enzyme during catalysis. Product interactions have received very little attention. CYP101 is the only P450 in which X-ray and spectroscopic data on all three structures, the substrate-free, camphor-bound and the 5-exo-OHcamphor-bound are available. The substrate-free CYP101 is low spin and six-coordinated with a water molecule ligated at the L(6) position of the iron. The substrate camphor binds to Site I, and releases the L(6) water despite its inability to bind to this site, indicating that Site I binding can inhibit Site II ligation. The product 5-exo-OHcamphor in addition to binding to Site I, binds to Site II through its -OH group forming Fe-O bond, resulting in the low spin six-coordinated complex. New temperature-jump relaxation kinetic data indicating that Site II ligation inhibits Site I binding are presented. It appears that the Site I and Site II function as interacting sub-sites. The inhibitory allosteric interactions between the two sub-sites are also reflected in the data on binding of the substrate camphor (S) in the presence of the product 5-exo-OH camphor (P) to CYP101 (E). The data are in accordance with the two-site model involving the ternary complex ESP. The affinity of the substrate to the product-bound enzyme as well as the affinity of the product to the substrate-bound enzyme decreased with increase in product concentration, which is consistent with mixed inhibition indicative of inhibitory allosteric interactions between the two sub-sites. Implications of these observations for coupling/uncoupling mechanisms are discussed in the light of the published findings consistent with the two-site behavior of the P450 active site. In addition, kinetic data indicating that the transient high spin intermediate may have to be taken into account for understanding how some P450s have been able to express appreciable hydroxylation activities in the absence of substrate-induced low to high spin transition, observable by the traditional static spectroscopy, are presented.     

Effect of alcohols on binding of camphor to cytochrome P450cam: spectroscopic and stopped flow transient kinetic studies

Addition of alcohols to cytochrome P450cam (CYP101) was shown to release the substrate camphor from the heme pocket of the enzyme. The release of the substrate was found to be caused both due to increased solubility of the substrate in solution in presence of alcohol and due to change in the tertiary structure of the active site of the enzyme. The far-UV CD and near-UV CD spectra reveal that addition of alcohols to cytochrome P450cam cause a small change in the secondary structural elements but a significant change in the tertiary structural organization of this enzyme. The CD spectra at the heme region at various concentrations of alcohols indicate a substantial change in the tertiary structural organization around the heme moiety too. The equilibrium constant associated with the binding of camphor to Cyt P450cam is strongly dependent on the concentration of alcohols and the corresponding free energy associated with the binding is found to scale linearly with the concentration of alcohols. Kinetic experiments on binding of camphor to Cyt P450cam show that both k(on) and k(off) rate constants are strongly affected by addition of alcohols suggesting that alcohol expel camphor out of the heme cavity of Cyt P450cam by affecting tertiary structure of Cyt P450cam as well as by modifying the solubility properties of camphor in aqueous medium.     

The roles of active site hydrogen bonding in cytochrome P-450cam as revealed by site-directed mutagenesis

The role of the active site hydrogen bond of cytochrome P-450cam has been studied utilizing a combination of site-directed mutagenesis and substrate analogues with altered hydrogen bonding capabilities. Cytochrome P-450cam normally catalyzes the regiospecific hydroxylation of the monoterpene camphor. The x-ray crystal structure of this soluble bacterial cytochrome P-450 (Poulos, T. L., Finzel, B. C., Gunsalus, I. C., Wagner, G. C., and Kraut, J. (1985) J. Biol. Chem. 260, 16122-16128) indicates a specific hydrogen bond between tyrosine 96 and the carbonyl moiety of the camphor substrate. The site-directed mutant in which tyrosine 96 has been changed to a phenylalanine and the substrate analogues thiocamphor and camphane have been used to probe this interaction in several aspects of catalysis. At room temperature, both the mutant enzyme with camphor and the wild type enzyme with thiocamphor bound result in 59 and 65% high-spin ferric enzyme as compared to the 95% high spin population obtained with native enzyme and camphor as substrate. The equilibrium dissociation constant is moderately increased, from 1.6 microM for the wild type protein to 3.0 and 3.3 microM for wild type-thiocamphor and mutant-camphor complexes, respectively. Camphane bound to cytochrome P-450cam exhibits a larger decrease in high spin fraction (45%) and a correspondingly larger KD (46 microM), suggesting that the carbonyl moiety of camphor plays an important steric role in addition to its interaction as a hydrogen bond acceptor. The absolute regioselectivity of the mutant enzyme, and of the wild type enzyme with thiocamphor, is lost resulting in production of several hydroxylated products in addition to the 5-exo-hydroxy isomer. Based on rates of NADH oxidation, comparison of the substrate specificity for these systems (kcat/KD) indicates a 5- and 7-fold decrease in specificity for the mutant enzyme and thiocamphor-wild type complex, respectively. The replacement of the cytochrome P-450cam active site tyrosine with phenylalanine does not affect the branching ratio of monooxygenase versus oxidase chemistry or peroxygenase activity (Atkins, W.M., and Sligar, S.G. (1987) J. Am. Chem. Soc. 109, 3754-3760).     

Proximal cysteine residue is essential for the enzymatic activities of cytochrome P450cam.

To investigate the functional and structural roles of the proximal thiolate ligand in cytochrome P450cam, we prepared the C357H mutant of the enzyme in which the axial cysteine residue (Cys357) was replaced with a histidine residue. We obtained the unstable C357H mutant by developing a new preparation procedure involving in vitro folding of P450cam from the inclusion bodies. The C357H mutant in the ferrous-CO form exhibited the Soret peak at 420 nm and the Fe-CO stretching line at 498 cm-1, indicating a neutral histidine residue as the axial ligand. However, another internal ligand is coordinated to the heme iron as the sixth ligand in the ferric and ferrous forms of the C357H mutant, suggesting the collapse of the substrate-binding site. The C357H mutant showed no catalytic activity for camphor hydroxylation and the reduced heterolytic/homolytic ratio of the O-O bond scission in the reaction with cumene hydroperoxide. The present observations indicate that the thiolate coordination in P450cam is important for the construction of the heme pocket and the heterolysis of the O-O bond.