The 2.6-A crystal structure of Pseudomonas putida cytochrome P-450

The crystal structure of Pseudomonas putida cytochrome P-450cam in the ferric, camphor bound form has been determined and partially refined to R = 0.23 at 2.6 A. The single 414 amino acid polypeptide chain (Mr = 45,000) approximates a triangular prism with a maximum dimension of approximately 60 A and a minimum of approximately 30 A. Twelve helical segments (A through L) account for approximately 40% of the structure while antiparallel beta pairs account for only approximately 10%. The unexposed iron protoporphyrin IX is sandwiched between two parallel helices designated the proximal and distal helices. The heme iron atom is pentacoordinate with the axial sulfur ligand provided by Cys 357 which extends from the N-terminal end of the proximal (L) helix. A substrate molecule, 2-bornanone (camphor), is buried in an internal pocket just above the heme distal surface adjacent to the oxygen binding site. The substrate molecule is held in place by a hydrogen bond between the side chain hydroxyl group of Tyr 96 and the camphor carbonyl oxygen atom in addition to complementary hydrophobic contacts between the camphor molecule and neighboring aliphatic and aromatic residues. The camphor is oriented such that the exo-surface of C5 would contact an iron bound, "activated" oxygen atom for stereoselective hydroxylation.     

Crystal structures of metyrapone- and phenylimidazole-inhibited complexes of cytochrome P-450cam

The crystal structures of metyrapone- and 1-, 2-, and 4-phenylimidazole-inhibited complexes of cytochrome P-450cam have been refined to a nominal resolution of 2.1 A and compared with the 1.63-A camphor-bound structure. With the exception of 2-phenylimidazole, each of the inhibitors forms an N-Fe bond with the heme iron atom while part of the inhibitor sits in the camphor-binding pocket. In the 2-phenylimidazole complex, a water molecule or hydroxide ion coordinates with the heme iron atom while the inhibitor binds in the camphor pocket adjacent to the aqua ligand. Each of the inhibitors forces the central region of helix I that forms part of the O2 binding pocket to move away from the inhibitor, with the exception of 2-phenylimidazole where the helix moves in toward the inhibitor. In addition, the Tyr-96 region, which provides specific contact points with the substrate, is perturbed, although to varying degrees with each inhibitor. These perturbations include large, localized changes in Debye-Waller or temperature factors, indicative of changes in dynamical fluctuations. The largest inhibitor, metyrapone, causes the fewest changes, while 2-phenylimidazole binding causes the largest, especially in helix I. The large 2-phenylimidazole-induced movement of helix I can be rationalized on the basis of the inhibitor imidazole group's hydrogen-bonding requirements.     

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.     

Structural evidence for a functionally relevant second camphor binding site in P450cam: model for substrate entry into a P450 active site

P450cam has long served as a prototype for the cytochrome P450 (CYP) gene family. But, little is known about how substrate enters its active site pocket, and how access is achieved in a way that minimizes exposure of the reactive heme. We hypothesize that P450cam may first bind substrate transiently near the mobile F-G helix that covers the active site pocket. Such a two-step binding process is kinetically required if P450cam rarely populates an open conformation-as suggested by previous literature and the inability to obtain a crystal structure of P450cam in an open conformation. Such a mechanism would minimize exposure of the heme by allowing P450cam to stay in a closed conformation as long as possible, since only brief flexing into an open conformation would be required to allow substrate entry. To test this model, we have attempted to dock a second camphor molecule into the crystal structure of camphor-bound P450cam. The docking identified only one potential entry site pocket, a well-defined cavity on the F-helix side of the F-G flap, 16 A from the heme iron. Location of this entry site pocket is consistent with our NMR T1 relaxation-based measurements of distances for a camphor that binds in fast exchange (active site camphor is known to bind in slow exchange). Presence of a second camphor binding site is also confirmed with [(1)H-(13)C] HSQC titrations of (13)CH3-threonine labeled P450cam. To confirm that camphor can bind outside of the active site pocket, (13)CH3-S-pyridine was bound to the heme iron to physically block the active site, and to serve as an NMR chemical shift probe. Titration of this P450cam-pyridine complex confirms that camphor can bind to a site outside the active site pocket, with an estimated Kd of 43 microM. The two-site binding model that is proposed based on these data is analogous to that recently proposed for CYP3A4, and is consistent with recent crystal structures of P450cam bound to tethered-substrates, which force a partially opened conformation.     

Formation, crystal structure, and rearrangement of a cytochrome P-450cam iron-phenyl complex

Cytochrome P-450cam reacts with phenyldiazene (PhN = NH), or less efficiently with phenylhydrazine, to give a catalytically inactive complex with an absorption maximum at 474 nm. The prosthetic group extracted anaerobically from the inactivated protein has the spectroscopic properties of a sigma phenyl-iron complex and rearranges, on exposure to air and acid, to an approximately equal mixture of the four N-phenylprotoporphyrin IX regioisomers. The crystal structure of the intact protein complex, refined at 1.9-A resolution to an R factor of 20%, confirms that the phenyl group is directly bonded through one of its carbons to the iron atom. The phenyl ring is tilted from the heme normal by about 10 degrees in the opposite direction from that in which carbon monoxide tilts when bound to P-450cam. Camphor, the natural substrate for P-450cam, is larger than a phenyl group and hydrogen bonds to Tyr 96, the only hydrophilic residue near the active site. Electron density in the active site in addition to that contributed by the phenyl group suggests that two water molecules occupy part of the camphor binding site but are not within hydrogen-bonding distance of Tyr 96. As observed in a previous crystallographic study of inhibitor-P-450cam complexes [Poulos, T.L., & Howard, A.J. (1987) Biochemistry 26, 8165-8174], there are large changes in both the atomic positions and mobilities of the residues in the proposed substrate access channel region of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Resonance Raman investigations of Escherichia coli-expressed Pseudomonas putida cytochrome P450 and P420.

High-resolution resonance Raman spectra of the ferric, ferrous, and carbonmonoxy (CO)-bound forms of wild-type Escherichia coli-expressed Pseudomonas putida cytochrome P450cam and its P420 form are reported. The ferric and ferrous species of P450 and P420 have been studied in both the presence and absence of excess camphor substrate. In ferric, camphor-bound, P450 (mos), the E. coli-expressed P450 is found to be spectroscopically indistinguishable from the native material. Although substrate binding to P450 is known to displace water molecules from the heme pocket, altering the coordination and spin state of the heme iron, the presence of camphor substrate in P420 samples is found to have essentially no effect on the Raman spectra of the heme in either the oxidized or reduced state. A detailed study of the Raman and absorption spectra of P450 and P420 reveals that the P420 heme is in equilibrium between a high-spin, five-coordinate (HS,5C) form and low-spin six-coordinate (LS,6C) form in both the ferric and ferrous oxidation states. In the ferric P420 state, H2O evidently remains as a heme ligand, while alterations of the protein tertiary structure lead to a significant reduction in affinity for Cys(357) thiolate binding to the heme iron. Ferrous P420 also consists of an equilibrium between HS,5C and LS,6C states, with the spectroscopic evidence indicating that H2O and histidine are the most likely axial ligands. The spectral characteristics of the CO complex of P420 are found to be almost identical to those of a low pH of Mb. Moreover, we find that the 10-ns transient Raman spectrum of the photolyzed P420 CO complex possesses a band at 220 cm-1, which is strong evidence in favor of histidine ligation in the CO-bound state. The equilibrium structure of ferrous P420 does not show this band, indicating that Fe-His bond formation is favored when the iron becomes more acidic upon CO binding. Raman spectra of stationary samples of the CO complex of P450 reveal VFe-CO peaks corresponding to both substrate-bound and substrate-free species and demonstrate that substrate dissociation is coupled to CO photolysis. Analysis of the relative band intensities as a function of photolysis indicates that the CO photolysis and rebinding rates are faster than camphor rebinding and that CO binds to the heme faster when camphor is not in the distal pocket.     

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.     

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.     

Mutagenesis of a single hydrogen bond in cytochrome P-450 alters cation binding and heme solvation

Cytochrome P-450cam, a monoxygenase responsible for the regiospecific hydroxylation of camphor, binds its substrate through complimentary van der Waals contacts and the formation of a single hydrogen bond between tyrosine 96 and the ketone group of camphor. Substrate association is positively regulated through the binding of a monovalent cation and the oxidation-reduction potential modulated by the spin state of the ferric heme controlled by water access to the sixth coordination site of the iron. Removal of this single hydrogen bond via site-directed mutagenesis of tyrosine 96 to phenylalanine 96 defines this aspect of the protein structure as responsible for the linkage between cation and substrate cooperativities, the degree of spin state conversion resulting from water access via macromolecule and substrate dynamics, and suggests a specific location for the cation binding site.     

A single mutation in cytochrome P450 BM3 induces the conformational rearrangement seen upon substrate binding in the wild-type enzyme

The multidomain fatty-acid hydroxylase flavocytochrome P450 BM3 has been studied as a paradigm model for eukaryotic microsomal P450 enzymes because of its homology to eukaryotic family 4 P450 enzymes and its use of a eukaryotic-like diflavin reductase redox partner. High-resolution crystal structures have led to the proposal that substrate-induced conformational changes lead to removal of water as the sixth ligand to the heme iron. Concomitant changes in the heme iron spin state and heme iron reduction potential help to trigger electron transfer from the reductase and to initiate catalysis. Surprisingly, the crystal structure of the substrate-free A264E heme domain mutant reveals the enzyme to be in the conformation observed for substrate-bound wild-type P450, but with the iron in the low-spin state. This provides strong evidence that the spin-state shift observed upon substrate binding in wild-type P450 BM3 not only is caused indirectly by structural changes in the protein, but is a direct consequence of the presence of the substrate itself, similar to what has been observed for P450cam. The crystal structure of the palmitoleate-bound A264E mutant reveals that substrate binding promotes heme ligation by Glu(264), with little other difference from the palmitoleate-bound wild-type structure observable. Despite having a protein-derived sixth heme ligand in the substrate-bound form, the A264E mutant is catalytically active, providing further indication for structural rearrangement of the active site upon reduction of the heme iron, including displacement of the glutamate ligand to allow binding of dioxygen.     

Chloroperoxidase: P-450 type absorption in the absence of sulfhydryl groups.

The oxidation state of the two half-cystine residues in the native ferric form of chloroperoxidase and in the reduced ferrous chloroperoxidase has been examined in order to evaluate the role of sulfhydryl groups as determinants of P-450 type spectra. M?ssbauer and optical spectroscopy studies indicate that the ferrous forms of P-450cam and chloroperoxidase have very similar or identical heme environments. Model studies have suggested that sulfhydryl groups may function as axial ligands for developing P-450 character. However, chemical studies involving both sulfhydryl reagents and amperometric titrations show that neither the ferric nor the chemically produced ferrous forms of chloroperoxidase contain a sulfhydryl group. These results rule out the hypothesis that sulfhydryl groups are unique components for P-450 absorption characteristics. The optical and electron paramagnetic resonance (EPR) spectra of the nitric oxide complex of chloroperoxidase have been obtained and compared to those of myoglobin, hemoglobin, and cytochrome c and horseradish peroxidase. The EPR spectrum of the NO-ferrous chloroperoxidase complex, which is similar to that of cytochrome P-450cam, does not show the extra nitrogen hyperfine structure which appears to be characteristic of those hemoproteins which have a nitrogen atom as an axial heme ligand.     

Proton coupling in the ligand-binding reaction of ferric cytochrome P-450 from Pseudomonas putida

Effects of pH on the ligand-binding reactions of ferric heme in cytochrome P-450 from Pseudomonas putida (camphor 5-monooxygenase, EC 1.14.15.1) were studied by using cyanide, N-methylimidazole, pyridine, and ethylisocyanide as ligands. In all cases, affinity of the ferric heme for the ligand was found to increase as pH of the medium was raised from around 6 to 9. Depending on the ligand, the increase was 10- to 1000-fold and the shapes of their pH-affinity curves were remarkably different. Analyses such pH profiles disclosed the presence of a dissociable group in the enzyme with a pK value of approximately 9.5 and that its ionization greatly enhanced the affinity of the heme for ligands. When a dissociable ligand such as hydrogen cyanide and N-methylimidazole was used, the dissociated form of the ligand had a higher affinity toward the heme than the undissociated form. The shapes of the pH-affinity curves were successfully simulated as overlapping curves of ionization reactions of the ligand and the dissociable group. In addition, size of the ligand molecule was shown to be also important in the binding reaction: relatively large molecules such as pyridine, ethylisocyanide, and N-methylimidazole bound to the enzyme in a competitive manner against d-camphor concentration, whereas the binding of a smaller molecule such as cyanide was inhibited by the substrate in a noncompetitive manner. On the basis of these findings, control mechanisms for the ligand-binding reactions of the cytochrome P-450 from P. putida are discussed.     

Ligand binding studies of engineered cytochrome P-450d wild type, proximal mutants, and distal mutants

Interactions of various axial ligands with cytochrome P-450d wild type, proximal mutants (Lys453Glu, Ile460Ser), and putative distal mutants (Glu318Asp, Thr319Ala, Thr322Ala) expressed in yeast were studied with optical absorption spectroscopy. P-450d wild type and all five mutants were purified essentially as the high-spin form, but the putative distal mutants contained about 5% low-spin form. Bindings of metyrapone and 4-phenylimidazole to the wild type and all mutants formed nitrogen-bound low-spin forms. In contrast, binding of 2-phenylimidazole to the wild type and most of mutants formed oxygen-bond low-spin forms except for the mutant Glu318Asp in which the nitrogen-bound low-spin form was formed. By analogy with the distal structure of P-450cam, it was thus suggested that Glu318 of P-450d, which corresponds with Asp251 of P-450cam, somehow interacts with 2-phenylimidazole over the heme plane. Addition of 1-butanol and acetanilide, a substrate of P-450d, to the wild type and mutants caused the spin change to the low-spin form. The order of dissociation constants of these oxygen ligands to P-450d was wild type greater than proximal mutants greater than putative distal mutants. Spectral analyses showed that the binding of acetanilide is the same as that of another substrate, 7-ethoxycoumarin, in the putative distal mutants but is not the same in the wild type and proximal mutants. From these findings together with other spectral data, it was suggested that the region from Glu318 to Thr322 is located at the distal region of the heme in membrane-bound P-450d as suggested from the X-ray crystal structure of water-soluble P-450cam and amino acid alignments of P-450s.     

Resonance Raman investigations of chloroperoxidase, horseradish peroxidase, and cytochrome c using Soret band laser excitation.

Resonance Raman spectra of the heme protein chloroperoxidase in its native and reduced forms and complexed with various small ions are obtained by using laser excitation in the Soret region (350-450 nm). Additionally, Raman spectra of horseradish peroxidase, cytochrome P-450cam, and cytochrome c, taken with Soret excitation, are presented and discussed. The data support previous findings that indicate a strong analogy between the active site environments of chloroperoxidase and cytochrome P-450cam. The Raman spectra of native chloroperoxidase are found to be sensitive to temperature and imply that a high leads to low spin transition of the heme iron atom takes place as the temperature is lowered. Unusual peak positions are also found for native and reduced chloroperoxidase and indicate a weakening of porphyrin ring bond strengths due to the presence of a strongly electron-donating axial ligand. Enormous selective enhancements of vibrational modes at 1360 and 674 cm-1 are also observed in some low-spin ferrous forms of the enzyme. These vibrational frequencies are assigned to primary normal modes of expansion of the prophyrin macrocycle upon electronic excitation.     

The P450cam G248E mutant covalently binds its prosthetic heme group

Previous studies on mammalian peroxidases and cytochrome P450 family 4 enzymes have shown that a carboxylic group positioned close to a methyl group of the prosthetic heme is required for the formation of a covalent link between a protein carboxylic acid side chain and the heme. To determine whether there are additional requirements for covalent bond formation in the P450 enzymes, a glutamic acid or an aspartic acid has been introduced into P450(cam) close to the heme 5-methyl group. Spectroscopic and kinetic studies of the resulting G248E and G248D mutants suggest that the carboxylate group coordinates with the heme iron atom, as reported for a comparable P450(BM3) mutant [Girvan, H. M., Marshall, K. R., Lawson, R. J., Leys, D., Joyce, M. G., Clarkson, J., Smith, W. E., Cheesman, M. R., and Munro, A. W. (2004) J. Biol. Chem. 279, 23274-23286]. The two P450(cam) mutants have low catalytic activity, but in contrast to the P450(BM3) mutant, incubation of the G248E (but not G248D) mutant with camphor, putidaredoxin, putidaredoxin reductase, and NADH results in partial covalent binding of the heme to the protein. No covalent attachment is observed in the absence of camphor or any of the other reaction components. Pronase digestion of the G248E P450(cam) mutant after covalent attachment of the heme releases 5-hydroxyheme, establishing that the heme is covalently attached through its 5-methyl group as predicted by in silico modeling. The results establish that a properly positioned carboxyl group is the sole requirement for autocatalytic formation of a heme-protein link in P450 enzymes, but also show that efficient covalent binding requires placement of the carboxyl close to the methyl but in a manner that prevents strong coordination to the iron atom.     

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.     

Electron-conformational interactions at the active site of reduced bacterial cytochrome P450cam induced by a substrate and analysis of the electron structure of heme]

Magnetic circular dichroism (MCD) spectra in the Soret region (360-480 nm) of camphor-free and camphor-bound reduced bacterial cytochrome P450cam from Pseudomonas putida were recorded and analysed in the temperature range from 2 K to 290 K. The temperature dependences of the MCD intensity are qualitatively changed by binding of substrate to the enzyme. In the absence of camphor the linear increase of the MCD intensity with 1/T at T < 4.2 K gives evidence for degeneracy or near degeneracy of the ground electronic state. In the presence of substrate the degeneracy is removed and temperature profiles show saturation behaviour at T < 4.2 K and wavelength dependence of their high-temperature parts. The temperature profiles for the long-wavelength region of the Soret band have a maximum approximately at 15 K, whereas the MCD intensity increases in a monotonous manner up to saturation in the short-wavelength region. The wavelength dependence of temperature profiles gives evidence for the co-existence of two different forms of substrate-bound reduced P450cam. The following conclusions were obtained from a theoretical analysis of the temperature profiles. In the absence of substrate there are very small if any rhombic distortions at the heme iron, and a parameter D of axial zero-field splitting is negative (D = -8.3 cm-1 and -6.2 cm-1 for P450cam and P450LM2, respectively). In the presence of substrate the two forms of reduced P450cam have positive parameters D but of different values (D1 = 12 cm-1 and D2 = 28 cm-1), and there are large rhombic distortions at the heme iron. More than two-fold difference between the D values made it possible to isolate temperature-dependent contributions of the two enzyme forms from the total MCD spectra and to simulate the alterations of the MCD spectra with temperature for reduced P450cam in the presence of substrate. Taking into account the drastic effect of substrate binding on the ground electronic state of reduced P450cam one can suggest that substrate binding induces the transition of enzyme from an inactive to an active state.     

Crystal structures of cytochrome P-450CAM complexed with camphane, thiocamphor, and adamantane: factors controlling P-450 substrate hydroxylation

X-ray crystal structures have been determined for complexes of cytochrome P-450CAM with the substrates camphane, adamantane, and thiocamphor. Unlike the natural substrate camphor, which hydrogen bonds to Tyr96 and is metabolized to a single product, camphane, adamantane and thiocamphor do not hydrogen bond to the enzyme and all are hydroxylated at multiple positions. Evidently the lack of a substrate-enzyme hydrogen bond allows substrates greater mobility in the active site, explaining this lower regiospecificity of metabolism as well as the inability of these substrates to displace the distal ligand to the heme iron. Tyr96 is a ligand, via its carbonyl oxygen atom, to a cation that is thought to stabilize the camphor-P-450CAM complex [Poulos, T. L., Finzel, B. C., & Howard, A. J. (1987) J. Mol. Biol. 195, 687-700]. The occupancy and temperature factor of the cationic site are lower and higher, respectively, in the presence of the non-hydrogen-bonding substrates investigated here than in the presence of camphor, underscoring the relationship between cation and substrate binding. Thiocamphor gave the most unexpected orientation in the active site of any of the substrates we have investigated to date. The orientation of thiocamphor is quite different from that of camphor. That is, carbons 5 and 6, at which thiocamphor is primarily hydroxylated [Atkins, W. M., & Sligar, S. G. (1988) J. Biol. Chem. 263, 18842-18849], are positioned near Tyr96 rather than near the heme iron. Therefore, the crystallographically observed thiocamphor-P-450CAM structure may correspond to a nonproductive complex. Disordered solvent has been identified in the active site in the presence of uncoupling substrates that channel reducing equivalents away from substrate hydroxylation toward hydrogen peroxide and/or "excess" water production. A buried solvent molecule has also been identified, which may promote uncoupling by moving from an internal location to the active site in the presence of highly mobile substrates.     

Heme-pocket-hydration change during the inactivation of cytochrome P-450camphor by hydrostatic pressure.

Hydrostatic pressure has been used to convert cytochrome P-450camphor to cytochrome P-420. The latter is an inactivated but soluble and undenaturated form of cytochrome P-450camphor. Using camphor analogues as probes of the active site we show that the inactivation volume change is directly correlated to the initial degree of hydration of the heme pocket. The values range between -73 ml/mol and -197 ml/mol [Di Primo, C., Hui Bon Hoa, G., Douzou, P. & Sligar, S. G. (1990) Eur. J. Biochem. 193, 383-386] for a totally hydrated (substrate-free, low-spin, six coordinated heme iron) and a non-hydrated (camphor-bound, high-spin, five coordinated heme iron) heme pocket. These results suggest that the larger value, -197 ml/mol, for the inactivation volume change is due to a hydration change of the heme pocket resulting from the displacement of the substrate during the compression and the subsequent entrance of water molecules. Similarly, the stability of the protein against compression is correlated with water accessibility to the active site. Increase in substrate mobility by loss of specific interactions with both regions of well defined secondary structure of cytochrome P-450camphor results in an increase of water accessibility and decrease of stability. Thus for camphor and adamantanone which strongly interact with the protein and exclude water from the active site [Poulos, T. L., Finzel, B. C. & Howard, A. J. (1987) J. Mol. Biol. 195, 687-700; Raag, R. & Poulos, T. L. (1989) Biochemistry 28, 917-922] the increase in stability compared to the free protein is roughly 30 kJ/mol at 20 degrees C. With smaller substrates such as norcamphor, which loosely fits into the active site and does not completely exclude water [Raag, R. & Poulos, T. L. (1989) Biochemistry 28, 917-922], the increase in stability is only 7 kJ/mol. Finally these results suggest that cytochrome P-420 induced by hydrostatic pressure is a unique form where the active site is hydrated and camphor is displaced from its binding site.     

Pseudomonas putida cytochrome P-450. The effect of complexes of the ferric hemeprotein on the relaxation of solvent water protons.

With pulsed nuclear magnetic resonance techniques, the effects of various complexes of ferric cytochrome P-450 on the relaxation rate of bulk solution water protons have been determined. For the camphor, metyrapone, and 4-phenylimidazole complexes, the experimental results are consistent with outer sphere relaxation effects. However, for the substrate-free enzyme, the magnitude and temperature dependence of the paramagnetic relaxation effects indicate the presence of exchangeable protons in the coordination sphere of the heme iron atom. The exchange rate (9.3 x 10(4) S-1 at 25 degrees) and the thermodynamic activation parameters for the exchange process are very similar to those of acid metmyoglobin and acid methemoglobin, suggesting that a water molecule, and not an amino acid residue of the protein, coordinates to the ferric cation of the enzyme in the absence of added substrate or ligands. From the equations appropriate for coordination sphere protons, the distance between these protons and the ferric heme cation was evaluated as 2.1 A, which further supports the interpretation. These experimental results demonstrate that the solvent accessibility of the ferric cation of substrate-free cytochrome P-450 is significantly reduced by the binding of substrate or nitrogenous ligands to the hemeprotein.