Circular dichroism studies of low-spin ferric cytochrome P-450CAM ligand complexes

Circular dichroism (CD) spectroscopy has been used to probe the active site of bacterial ferric cytochrome P-450CAM. The endogenous sixth ligand to the heme iron has been displaced by an extensive series of exogenous oxygen, nitrogen, sulfur and other neutral and anionic donor ligands in an attempt to examine systematically the steric and electronic factors that influence the coupling of the heme chromophore to its protein environment. General trends for each ligand class are reported and discussed. Both the wavelengths and the intensities of the CD bands vary with ligand type and structure. All but one of the complexes exhibit negative CD maxima in their delta and Soret bands. Comparison to ferric myoglobin-thiolate complexes indicates that the negative sign observed for the cytochrome P-450 spectra is not a property of the thiolate fifth ligand, but rather arises from a different interaction of the cytochrome P-450 heme with its protein environment. Complexes with neutral oxygen donors display CD spectra that most closely resemble the spectrum of the native low-spin enzyme. Hyperporphyrin (split Soret) cytochrome P-450 complexes with thiolates, phosphines and cyanide trans to cysteinate have complex CD spectra, reflecting the intrinsic non-degeneracy of the Soret pi pi transitions. The extensive work presented herein provides an empirical foundation for use in analyzing the interaction of heme chromophores with their protein surroundings, not only for the cytochrome P-450 monooxygenases but also for heme proteins in general.     

Spectroscopic investigations of ferric cytochrome P-450-CAM ligand complexes. Identification of the ligand trans to cysteinate in the native enzyme

An extensive series of ligand complexes of ferric cytochrome P-450-CAM has been examined by UV-visible absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopy in an attempt to identify the ligand trans to cysteinate in the six-coordinate resting state of the enzyme. Thus, the ligands used have been chosen to serve as models for coordination by potential endogenous amino acids and include alcohol, amide and carboxylate oxygen donors, amine, imidazole and indole nitrogen donors and disulfide, thioether, thiol, and thiolate sulfur donors. As this investigation has been by nature an empirical one, the conclusions are strengthened by the concurrent use of three different spectroscopic techniques. All of the complexes formed except those resulting from thiolate addition display spectroscopic properties that are broadly similar to those of low spin, six-coordinate P-450. Of the sulfur donor adducts, disulfide and thioether-bound P-450 have properties that are different enough in detail to distinguish them from native P-450. While the spectral features of the thiol-bound species and of low spin ferric P-450 are alike, the former are pH dependent due to interconversion to bound thiolate, whereas the latter display essentially no spectral changes with pH. Of the oxygen donor complexes, all but carboxylate have spectra that very closely match those of the resting enzyme. Adducts formed with most nitrogenous ligands, including several imidazole derivatives, exhibit spectra that are sufficiently different from native P-450 to exclude them as candidates for the sixth ligand. Interestingly, the spectral properties of a complex formed with an imidazole derivative having a bulky electron-withdrawing substituent in the alpha position are comparable to those native P-450 except for the line shape of the EPR spectrum. Previously published theoretical work suggests that the spectral differences seen between this imidazole derivative and the other examined are electronic and not steric in origin. As no similar electronic mechanism exists for the protein to reduce the electron-donating ability or histidine, it is felt that coordination of histidine in the sixth position of P-450 can be ruled out. In conclusion, close examination of all spectral data reveals that amino acid analog adducts of P-450-CAM with amides and, in particular, alcohols, produce spectra that almost exactly duplicate those of native P-450 and suggests that the ligand trans to cysteinate in the six-coordinate ferric enzyme has an oxygen donor atom.     

Models for ferric cytochrome P450. Characterization of hemin mercaptide complexes by electronic and ESR spectra.

Hemin coordinated with mercaptide sulfur as fifth ligand and various sixth ligands were investigated as models for cytochrome P450 in its native ferric low-spin state and its ligand complexes. Mixing the hemin with its ligands below -60 degrees C prevented the reduction of the hemin by mercaptide and made it possible to characterize each sample both by electronic and ESR spectra. Excess of mercaptide formed hemin-dimercaptide complexes with hyperporphyrin spectra with two Soret bands around 380 and 370 nm. The second mercaptide could be exchanged by other ligands with hydroxyl, phosphine, thioether, isocyanide, amine, imidazole, and pyridine groups. The comparison of these spectral data with cytochrome P450 substantiates mercaptide as the fifth ligand and makes a hydroxyl group a more likely candidate for the native sixth ligand than an imidazole group.     

Quantum chemical interpretation of the spectral properties of the CO and O2 complexes of hemoglobin and cytochrome P-450

The electronic transitions of CO and O2 complexes of hemoglobin and cytochrome P-450 were calculated using a PPP method extended for metal complexes. The calculations show that the unusual spectral properties of cytochrome P-450 are very sensitive to the iron-sulfur bond distance. It is suggested from these calculations that for the conversion of cytochrome P-450 to cytochrome P-420 an increase of the iron-sulfur bond distance of only about 0.2 A is sufficient. The anomalous Soret band of the CO complex as well as the normal Soret band of the O2 complex of cytochrome P-450 are explicable assuming a mercaptide sulfur as fifth ligand.     

Binding of thiols to microsomal cytochrome P-450.

Lipophilic thiol compounds interact spectrally with liver microsomes from phenobarbital-pretreated rats by formation of unusual optical difference spectra with peaks at 378, 471, 522 and 593 nm in the oxidized state. The binding kinetics were biphasic. The EPR spectrum of cytochrome P-450 was slightly modified but the magnitude of the low-spin signal was unchanged. n-Octanethiol competitively displaced metyrapone and n-octane from the active site of cytochrome P-450. Other thiols behaved similarly with variations in the magnitude and the affinity of the binding process. Tertiary thiols caused the formation of the high-spin cytochrome P-450 substrate complex, and model studies with myoglobin revealed that steric hindrance prevented the liganding of the tertiary thiol group to the ferric cytochrome P-450. Addition of thiols to dithionite reduced microsomes resulted in relatively small spectral changes with maxima at 449 nm typical for ligand complexes of the ferrous cytochrome. It was concluded that lipophilic thiols can be bound as ligands by at least two species of oxidized cytochrome P-450 which represent, however, not more than about one fifth of the total cytochrome P-450 content in liver microsomes from phenobarbital-pretreated rats.     

Electron paramagnetic resonance investigations of exogenous ligand complexes of low-spin ferric chloroperoxidase: further support for endogenous thiolate ligation to the heme iron.

Previous spectroscopic studies of chloroperoxidase have provided evidence for endogenous thiolate sulfur donor ligation to the central heme iron of the enzyme. This conclusion is further supported by recent DNA sequence data which revealed the existence of a third cysteine residue (in addition to a disulfide pair detected earlier) in the protein available for coordination to the heme iron. Thus, chloroperoxidase shares many spectroscopic properties with cytochrome P-450, the only other known thiolate-ligated heme protein. Surprisingly, a previous electron paramagnetic resonance (EPR) study of low-spin ferric chloroperoxidase-ligand complexes (Hollenberg, P.F., Hager, L.P., Blumberg, W.E. and Peisach, J. (1980) J. Biol. Chem. 255, 4801-4807) was unable to provide clear support for the presence of a thiolate ligand, although sulfur coordination was implicated. This was, in part, because an insufficient number of complexes was examined. In this work, we have significantly expanded upon the previous EPR study by using an extensive variety of over twenty exogenous ligands including carbon, nitrogen, oxygen, phosphorus and sulfur donors. Crystal field analysis, using the procedure of Blumberg and Peisach, of the present data in comparison with data for analogous complexes of cytochrome P-450-CAM, thiolate-ligated heme model systems, and myoglobin, is clearly indicative of endogenous thiolate ligation for chloroperoxidase. In addition, the UV-visible absorption and EPR spectral data suggest that a carboxylate ligand is a possible candidate for the endogenous sixth ligand to the heme iron that is responsible for the reversible conversion of ferric chloroperoxidase from high-spin to low-spin at low temperatures (less than 200 K).     

The diverse spectroscopic properties of ferrous cytochrome P-450-CAM ligand complexes

The UV-visible absorption, magnetic circular dichroism (MCD) and CD spectral characteristics of a variety of low spin ferrous P-450-ligand complexes have been carefully determined in order to establish whether all such complexes are hyperporphyrins as previously suggested in the literature. Two general spectral classes are found to occur. Complexes in the first class are, indeed, hyperporphyrin in nature, with pi-acceptor ligands such as CO, NO, phosphine, nitrosoalkanes and isocyanides trans to cysteinate. Individual, but minor, variations in the spectral properties of the hyperporphyrins suggest that subclasses exist, wherein the nature of the trans ligand to thiolate affects the orbital overlap pattern and thus the observed spectra. Adducts in the second spectral class, which have sigma-donor nitrogen and sulfur ligands, also have the red-shifted Soret absorption maximum but are spectrally distinct in all other respects from the hyperporphyrins. Comparison of the MCD spectra of the second category to those of ferrous cytochromes b5, c, and P-420 suggests that the axial cysteinate ligand is still present in the nonhyper ferrous P-450 species. Thus, the combination of a strongly electron-donating cysteinate ligand and a trans sigma-donor, not the orbital mixing mechanism, is most likely the origin of the red-shifted Soret absorption maximum of nonhyper ferrous P-450 ligand complexes. Further, the nature of the total electronic interactions between both axial ligands and the heme iron of ferrous P-450 and not solely the cysteinate ligand determines whether the ligand complexes will be of the hyper or nonhyperporphyrin category. These findings are strengthened by the simultaneous use of three different spectroscopic techniques; together they provide a more detailed explanation for the unusual spectroscopic properties of cytochrome P-450.     

Spectral properties of a novel cytochrome P-450 of a Saccharomyces cerevisiae mutant SG1. A cytochrome P-450 species having a nitrogenous ligand trans to thiolate

An altered cytochrome P-450 (SG1 P-450) was partially purified from Saccharomyces cerevisiae mutant SG1 which is defective in lanosterol 14 alpha-demethylation. Oxidized SG1 P-450 showed a Soret peak at 422 nm and the alpha peak was lower than the beta peak. This spectrum was considerably different from those of known low-spin P-450s, indicating a unique ligand structure of SG1 P-450. The absorption spectrum of ferric SG1 P-450 was superimposable on that of the imidazole complex of ferric P-450, suggesting the presence of a nitrogenous ligand such as histidine of the apoprotein at the 6th coordination position. SG1 P-450 was immunochemically indistinguishable from cytochrome P-450 of S. cerevisiae catalyzing lanosterol 14 alpha-demethylation (P-45014DM) but had no lanosterol 14 alpha-demethylase activity.     

Low- and ultralow-temperature magnetic circular dichroism studies of reduced cytochromes P-450-LM2 and P-420-LM2 and of photo-products of their co-complexes. The spin-state and axial ligation of heme iron

MCD spectra of reduced cytochromes P-450 and P-420 have been recorded in the spectral region 350-800 nm at temperatures 4.2-290 K and were compared with the respective low-temperature photolysed CO-complexes at 4.2 K. The MCD data are consistent with the suggestions that: the heme iron is high-spin in the reduced proteins and in the photolysed species; mercaptide is the protein-derived ligand of the heme iron in the reduced cytochrome P-450, as well as in its CO-complex; imidazole of histidine is the fifth ligand of the heme iron both in the reduced P-420 and its CO-complex; structural changes in the heme iron coordination sphere occur at CO-binding.     

Thermodynamic studies of the protein-protein interactions between cytochrome P-450 and cytochrome b5. Evidence for a central role of the cytochrome P-450 spin state in the coupling of substrate and cytochrome b5 binding to the terminal hemoprotein

The interactions between purified rat hepatic microsomal cytochrome P-450 and the type I ligands benzphetamine and cytochrome b5 have been studied in the presence of phospholipid using difference spectrophotometry. Cytochrome b5 was shown to interact with cytochrome P-450 to form a tight 1:1 complex (Kd = 275 nM), in which the proportion of high spin cytochrome P-450 was increased from 7 to 30%. The presence of saturating cytochrome b5 was shown to cause a decrease in the apparent Kd for benzphetamine binding from 111 microM to 40 microM. Likewise, the presence of benzphetamine was shown to cause a decrease in the apparent dissociation constant for cytochrome b5 binding to cytochrome P-450 (Kd = 90 nM). The above interactions were resolved into the basic equilibria inter-relating the various ligation states of the hemoprotein in an energetically closed eight-state free energy coupling model and the relative magnitudes of the microequilibria were analyzed to determine the degree of coupling of the interactions between cytochrome P-450 and both benzphetamine and cytochrome b5. Consequently, the spin state changes in cytochrome P-450 induced by benzphetamine and cytochrome b5 binding were shown to arise because these ligands interact 7 and 4 times more tightly with high spin cytochrome P-450, respectively. Furthermore, the data revealed that these ligands interact at independent sites on cytochrome P-450. Thus the effects of cytochrome b5 upon benzphetamine binding and vice versa were rationalized simply in terms of an increase in the proportion of a high spin (high affinity) conformation of cytochrome P-450 brought about by pre-equilibration with the effector ligand, with the intrinsic binding affinities of the two ligands for the low or high spin states remaining relatively unaltered. The thermodynamic parameters associated with the interactions between cytochrome P-450 and cytochrome b5, determined from the temperature dependence of these interactions, revealed that these protein interactions are entropy driven and probably occur by a hydrophobic mechanism.     

Intramolecular photoredox reactions in iron(III) cytochrome c and its azide derivative

The irradiation of deaerated solutions of horse heart cytochrome c causes the reduction of Fe(III) to Fe(II). The dependence of the photoreaction quantum yield on pH shows that the photoreactive species is a form of cytochrome c which contains methionine-80 and histidine-18 as heme ligands. The primary photochemical event consists of an electron transfer from the sulphur of methionine- 80 to iron. The re-oxidation of the photochemically obtained Fe(II) protein gives a Fe(III) cytochrome which exhibits a typical low-spin absorption spectrum, lacking the 695-nm band and indicating that a strong field ligand, other than methionine-80, coordinates to the sixth binding site of the heme iron. Spectrophotometric titration of the photochemically modified Fe(III) cytochrome shows that histidine- 18 remains bound in the fifth position.The substitution of methionine-80 with the more oxidizable azide ligand increases the efficiency of the intramolecular electron transfer. Azide radicals, detected by spin-trapping ESR technique, are formed in the primary act. Visible-UV spectral data indicate that histidine-18 and methionine-80 occupy the fifth and sixth position, respectively, in the photoreaction product. All the results obtained correlate well with those previously obtained in investigations concerning the photoredox behavior of iron porphyrin complexes.     

An anomaly in the resonance Raman spectra of cytochrome P-450cam in the ferrous high-spin state.

Resonance Raman spectra of cytochrome P-450cam (P-450cam) and its enzymatically inactive form (P-420) in various oxidation and spin states were measured for the first time. The Raman spectrum of reduced P-450cam was unusual in the sense that the "oxidation-state marker" appeared at an unexpectedly lower frequency (1346 cm-1) in comparison with those of other reduced hemoproteins (approximately 1355-approximately 1365 cm-1), whereas that of oxidized P-450cam was located at a normal frequency. This anomaly in the Raman spectrum of reduced P-450cam can be explained by assuming electron delocalization from the fifth ligand, presumably a thiolate anion, to the antibonding pi orbital of the porphyrin ring. The corresponding Raman line of reduced P-420 appeared at a normal frequency (1360 cm-1), suggesting a status change or replacement of the fifth ligand upon conversion from P-450cam to P-420. The Raman spectrum of reduced P-450cam-metyrapone complex was very similar to that of ferrous cytochrome b5.     

The role of 5-aminolaevulinate synthase, haem oxygenase and ligand formation in the mechanism of maintenance of cytochrome P-450 concentration in hepatocyte culture.

The present work shows that the ability of pyridines e.g. metyrapone, to maintain the cytochrome P-450 concentration in cultured hepatocytes is not due to their ability to alter the 5-aminolaevulinate synthase and haem oxygenase activities of the hepatocytes. Since ligands such as metyrapone will prevent the cobalt-mediated loss of hepatic cytochrome P-450 in rats, the hypothesis that ligand formation is the mechanism of maintenance of the cytochrome in hepatocyte culture was tested. The observation that non-pyridine ligands will maintain the cytochrome P-450 concentration supports this hypothesis.     

Replacement of putative axial ligands of heme iron in yeast cytochrome c1 by site-directed mutagenesis

The His-44 and Met-164 residues of yeast cytochrome c1 are evolutionally conserved and regarded as heme axial ligands bonding to the fifth and sixth coordination sites of the heme iron, which is directly involved in the electron transfer mechanism. Oligonucleotide-directed mutagenesis was used to generate mutant forms of cytochrome c1 of yeast having amino acid replacements of the putative axial ligands of the heme iron. When a cytochrome c1-deficiency yeast strain was transformed with a gene encoding the Phe-44, Tyr-44, Leu-164, or Lys-164 protein, none of these transformants could grow on the non-fermentable carbon source. These results suggest that the His-44 and Met-164 residues have a critical role in the function of cytochrome c1 in vivo, most probably as axial ligands of the heme iron. Further analysis revealed that the mutant yeast cells with the Phe-44, Tyr-44, or Leu-164 protein lacked the characteristic difference spectroscopic signal of cytochrome c1. However, in the Lys-164 mutant cells, partial recovery of the cytochrome c1 signal was observed. Moreover, the Lys-164 protein retained a low but significant level of succinate-cytochrome c reductase activity in vitro. The possibility that the nitrogen of Lys-164 served as the sixth heme ligand is discussed in comparison with cytochrome f of a photosynthetic electron-transfer complex, in which lysine has been proposed to be the sixth ligand.     

1H nuclear magnetic resonance and magnetic circular dichroism studies of ferric low-spin cytochrome P-450scc

400 MHz ¹H NMR of ferric low-spin cytochrome P-450_scc purified from bovine adrenal cortex was measured for the first time. As compared with ¹H NMR spectra of low-spin P-450_cam and metMb- mercaptan complexes, paramagnetic shifts of low-spin P-450_scc complexes were more divergent, suggesting that there is a subtle difference in the heme environment between P-450_scc and P-450_cam [1]. The paramagnetic shifts of low-spin complexes of P-450_scc caused by adding nitrogenous inhibitors, aminoglutethimide and metyrapone, were different from those caused by adding an intermediate, 20α-hydroxycholesterol, and a detergent, Tween 20 [2]. The paramagnetic shifts of the metMb-mercaptan complexes were convergent compared with those of ferric low-spin P-450_scc and P-450_cam, suggesting that the electronic character and/or the conformation of the internal thiolate ligand in P-450_scc and P-450_cam are different from those of the external thiolate ligand in metMb-thiolate complexes [3]. The paramagetic shifts of the metMb-mercaptan complexes were dependent on the electron donating factor of the alkyl group of the bound mercaptans [4].Magnetic CD(MCD) spectra of ferric low-spin P-450_scc, rabbit liver P-450 complexes and metMb- mercaptan complexes were also observed at various temperatures. The temperature dependences of the Soret MCD bands for the low-spin P-450 and metMb- mercaptan complexes were decidedly less pronounced than those for the low-spin metMb-CN? or imidazole complexes, suggesting that thiolate ligands markedly influence the Soret MCD band of the ferric low-spin complexes [1]. The suggestion described in [2] implied by the ¹H NMR study was reconfirmed from the temperature dependence study of the Soret MCD [2]. The temperature dependences of the Soret MCD bands for low-spin P-450 complexes having a non-nitrogenous ligand were more pronounced than for those having a nitrogenous ligand.     

Effects of cholesterol analogues and inhibitors on the heme moiety of cytochrome P-450scc: a resonance Raman study

Interactions of cholesterol analogues and inhibitors with the heme moiety of cytochrome P-450scc were examined by resonance Raman spectroscopy. The Raman spectra of ferric cytochrome P-450scc complexed with inhibitors such as cyanide, phenyl isocyanide, aminoglutethimide, and metyrapone were characteristic of low-spin state and were very similar. However, the effect of exchange of the sixth ligand from the oxygen atom (ferric low-spin state) to the nitrogen atom upon aminoglutethimide and metyrapone binding was seen as down-frequency shifts of the v3 band from 1503 to 1501 and 1502 cm-1, respectively, while cyanide and phenyl isocyanide binding caused an up-frequency shift of the v3 band to 1505 cm-1. The effects of cholesterol analogues [22(R)-hydroxycholesterol, 22(S)-hydroxycholesterol, 22-ketocholesterol, 20(S)-hydroxycholesterol, and 25-hydroxycholesterol] on a Fe2+-CO stretching frequency of cytochrome P-450scc in ferrous CO form were examined. The 22(R)-hydroxycholesterol complex could not give a clear Fe2+-CO stretching Raman band due to a strong photodissociability. 22(S)-Hydroxycholesterol and 25-hydroxycholesterol complexes gave the Raman bands at 487 and 483 cm-1, respectively, whereas 20(S)-hydroxycholesterol and 22-ketocholesterol complexes gave Fe2+-CO stretching frequencies (478 cm-1) almost identical with that without substrate (477 cm-1). These findings suggest the existence of the following physiologically important natures of the cytochrome P-450scc active site: (1) there is a strong steric interaction between heme-bound carbon monoxide and the 22(R)-hydroxyl group or the 22(R)-hydrogen of the steroid side chain and (2) the hydroxylation at the 20S position may cause a conformational change of the side-chain group relative to the heme.     

Complexes of trivalent oxygenated phosphorus compounds with cytochrome P-450 and cytochrome P-420: the origin of double Soret spectra.

Trivalent oxygenated phosphorus ligands include alkyl and aryl phosphites, (RO)3P, phosphonites, (RO)2PR, and phosphinites, ROPR2. All such compounds tested, with the exception of triphenyl phosphite, interact with ferrous cytochrome P-450 and its denatured form, cytochrome P-420, to produce complexes having two peaks in the Soret region of their optical difference spectra. Careful evaluation of these spectra indicate that they arise for different reasons for each of the two cytochromes. Clear evidence shows that cytochrome P-450 is not denatured by these ligands. The high affinity of these ligands for heme iron is indicated by small Ks values. The experimental results are used to substantiate a theory of the origin of microsomal double Soret spectra and the nature of the environments available for microsomal cytochromes P-450 and P-420.     

Models for coordination site of cytochrome P-450, characterization of hemin-thiolato complexes with S, O, and N donor ligands by electronic absorption and electron spin resonance spectra

Bisthiolato-hemin complexes exhibiting "two split Soret bands" at 370 and 460 nm, classified into "hyperporphyrin spectrum" was prepared with naturally occurring porphyrins (Fe(III)protoporphyrin IX and its dimethyl ester), thioglycolate esters, and tetramethylammonium hydroxide in organic solvents. The structure of the complexes was characterized by electronic absorption and electron spin resonance (ESR) spectrometries. These complexes were stable under air at room temperature, their apparent half-lives being about 30 min monitored by the intensities of the two Soret bands. Thus the bisthiolato-hemin complex containing thioglycolate ester was shown to be a model for the cytochrome P450(P450)-thiolato binding complex. Ligand exchange reactions of the bisthiolato-hemin complex with imidazole or methanol indicated that the intermediate species are stabilized as thiolato-hemin-imidazole or -methanol complexes. The latter intermediate complex was suggested to be a good model for low-spin ferric P450 as characterized by distinct beta- and alpha-bands at 530 and 560 nm, respectively, as well as a single Soret peak at approximately 410 nm. The result of the analysis on ESR g values and crystal field parameters for the bisthiolato-hemin, thiolato-hemin-imidazole, and thiolato-hemin-oxygen ligand complexes comparing with those for P450 itself and the ligand binding complexes revealed that the sixth ligand trans to the fifth thiolato ligand of the low-spin ferric P450 can be an oxygen atom of water molecule.     

Model of the coordination site for cobalt-substituted cytochrome P450cam

Cobalt-substituted cytochrome P450_cam was recently reconstituted by Wagner et al (J. Biol. Chem. 256, 6266, (1981)). A model of its coordination site was constructed to determine the mode of axial coordination of the native enzyme. Complexes were prepared from cobalt porphyrins (cobalt-protoporphyrin IX (CoPPIX), cobalt-meso-tetraphenylporphyrin, cobalt-γ-laurylpyridyl triphenylporphyrin, and cobalt-octaethylporphyrin), thioglycolate ester, and tetramethylammonium hydroxide in organic solvents. Complexes prepared in an organic solvent such as CHC1₃ under air at room temperature exhibited a stable Soret hyperporphyrin spectrum characterized by split Soret bands, especially like that of the thiol-Co-P450_cam complex Comparison of the spectra of the hyperporphyrin spectral complexes titrated with various types of alcohol and imidazole, with the spectrum of Co-P450_cam in the oxidized state support the idea that an axial thiolate at the fifth position and a hydroxyl group of alcohol at the sixth position of the heme form the coordination site of Co-P450_cam The CoPPIX-thiolate-ethanol complex retaining S⁻-Co(III)-OH coordination is thought to be a possible model of Co-P450_cam in the oxidized state.     

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.