Resonance Raman detection of bound dioxygen in cytochrome P-450cam

We have used resonance Raman spectroscopy and isotopic labeling techniques to unambiguously assign the dioxygen stretching frequency (vo-o) in the substrate-bound oxygenated complex of cytochrome P-450cam. The frequency found for Vo-o in the P-450cam system (1140 cm-1) is in remarkable agreement with recent studies of thiolate heme model compounds. The general features of the oxy-P-450cam Raman spectra are tabulated and comparisons are made with the oxy complexes of hemoglobin, myoglobin, and various model compounds. Most of the results are qualitatively explained by consideration of electron donation into the pi g (O2)/d pi (M) orbitals of the oxygenated complex (M = Fe or Co). It is also noted that the effect of the "extra" electron in the nitrogen base Co(II) oxy complexes, in some ways, parallels the effect of the lone pair electrons of thiolate in the oxy-P-450cam complex. This is evidenced by the enhanced resonance Raman activity of vo-o in both the Co(II) and P-450 systems as well as by the similarity of the vo-o frequencies.     

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.     

Comparative EPR study on high-spin ferric porphine complexes and cytochrome P-450 having rhombic character.

Comparative EPR studies were made on two high-spin Fe(III) porphine model systems and mammalian liver microsomal cytochromes P-450, all of which exhibit approximately the same degrees of rhombicity in their EPR spectra. Comparison of g values and linewidths as a function of temperature, and of the microwave power saturation demonstrated that EPR characteristics of P-450 are more similar to the Fe(III) porphines having the thiolate axial ligand than in the other model systems, the mixed crystals of Fe(III) porphine with the corresponding free base porphine, in which no thiolate ligand is involved. There is, however, a discrepancy between P-450 and the model thiolates with respect to the size of the zero-field parameter D. These observations indicate that P-450 heme has essential structural features in common with thiolates but the Fe-S bond of P-450 may be modified from its normal orientation in model thiolates, probably as a result of the constraints imposed by the protein structure.     

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.     

Spectral characterization of brain and macrophage nitric oxide synthases. Cytochrome P-450-like hemeproteins that contain a flavin semiquinone radical.

Nitric oxide (NO) is synthesized in mammals where it acts as a signal molecule for neurotransmission, vasorelaxation, and cytotoxicity. The NO synthases isolated from brain and cytokine-activated macrophages are FAD- and FMN-containing flavoproteins that display considerable sequence homology to NADPH-cytochrome P-450 reductase. However, the nature of their catalytic centers is unknown. We have found that both isoenzymes contain 2 mol of iron-protoporphyrin IX/mol of enzyme homodimer. The optical and EPR spectroscopic properties of the heme groups were found to be remarkably similar to those of high-spin cytochrome P-450. The heme iron in the resting NO synthase is ferric and five-coordinate with a cysteine thiolate as the proximal axial ligand. In addition, the EPR spectra of the resting NO synthases contained a free radical signal attributable to a bound flavin semiquinone that appeared to interact magnetically with the ferric heme iron. NO production was inhibited by carbon monoxide, implying a role for the heme groups in catalysis.     

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.     

Apoprotein formation and heme reconstitution of cytochrome P-450cam

Apoprotein suitable for heme reconstitution has been prepared by an acid/butanone extraction of cytochrome P-450cam at pH 2.5. Absorption spectra of apo-P-450cam indicate less than 2% residual holoenzyme. Four tryptophan residues per molecule were estimated from the aromatic absorbance region of denatured apoprotein. Heme-reconstituted holoprotein was purified in 30% yield to a specific activity equivalent to the native enzyme. Absorption and EPR spectra of 57Fe- and 54Fe-heme-enriched P-450cam reveal complete restoration of the native active site.     

Cobalt-substituted horseradish peroxidase.

Horseradish peroxidase can be reconstituted with cobalt porphyrin to give a cobaltic holoenzyme having physicochemical properties quite similar to those of the native ferric protein. The cobaltic protein (Co3+HRP) can be reduced to the cobaltous form (CoHRP), the analogue of ferroperoxidase and the reduced cobalt protein can bind O2 to form an analogue of oxyferroperoxidase (Compound III). Since both the CoHRP and oxy-CoHRP are EPR-visible, the cobalt has been used to probe the nature of the heme crevice in these two protein forms. The occurrence of a three-line 14N superhyperfine pattern in the spectrum of the former unambiguously shows that in the divalent state of the protein the proximal axial ligand is a nitrogenous base. The spectrum of the latter shows a uniquely large Aparallel(59Co) = 23.2 G. Although we confirm the reported failure of the Co3+HRP to catalyze peroxide-dependent oxidations of classical peroxidase substrates (Gjessing, E.C., and Sumner, J.B. (1942) Arch. Biochem. 1, 1), the oxy-CoHRP does undergo oxidation-reduction reactions analogous to those exhibited in the cytochrome P-450 catalytic cycle.     

Cytochrome P-450cam and putidaredoxin interaction during electron transfer.

Cytochrome P-450cam, the bacterial hemeprotein which catalyzes the 5-exo-hydroxylation of d-camphor, requires two electrons to activate molecular oxygen for this monooxygenase reaction. These two electrons are transferred to cytochrome P-450cam in two one-electron steps by the physiological reductant, putidaredoxin. The present study of the kinetics of reduction of cytochrome P-450cam by reduced putidaredoxin has shown that the reaction obeys first order kinetics with a rate constant of 33 s-1 at 25 degrees C with respect to: 1) the appearance of the carbon monoxide complex of Fe(II) cytochrome P-450cam; 2) the disappearance of the 645 nm absorbance band of high-spin Fe(III) cytochrome P-450cam; and 3) the disappearance of the g = 1.94 EPR signal of reduced putidaredoxin. This data was interpreted as indicative of the rapid formation of a bimolecular complex between reduced putidaredoxin Fe(III) cytochrome P-450cam. The existence of the complex was first shown indirectly by kinetic analysis and secondly directly by electron paramagnetic resonance spectroscopic analysis of samples which were freeze-quenched approximately 16 ms after mixing. The direct evidence for complex formation was the loss of the EPR signal of Fe(III) cytochrome P-450cam upon formation of the complex while the EPR signal of reduced putidaredoxin decays with the same kinetics as the appearance of Fe(II) cytochrome P-450. The mechanism of the loss of the EPR signal of cytochrome P-450 upon formation of the complex is not apparent at this time but may involve a conformational change of cytochrome P-450cam following complex formation.     

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.     

Engineering cytochrome c peroxidase into cytochrome P450: a proximal effect on heme-thiolate ligation.

In an effort to investigate factors required to stabilize heme-thiolate ligation, key structural components necessary to convert cytochrome c peroxidase (CcP) into a thiolate-ligated cytochrome P450-like enzyme have been evaluated and the H175C/D235L CcP double mutant has been engineered. The UV-visible absorption, magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectra for the double mutant at pH 8.0 are reported herein. The close similarity between the spectra of ferric substrate-bound cytochrome P450cam and those of the exogenous ligand-free ferric state of the double mutant with all three techniques support the conclusion that the latter has a pentacoordinate, high-spin heme with thiolate ligation. Previous efforts to prepare a thiolate-ligated mutant of CcP with the H175C single mutant led to Cys oxidation to cysteic acid [Choudhury et al. (1994) J. Biol. Chem. 267, 25656-25659]. Therefore it is concluded that changing the proximal Asp235 residue to Leu is critical in forming a stable heme-thiolate ligation in the resting state of the enzyme. To further probe the versatility of the CcP double mutant as a ferric P450 model, hexacoordinate low-spin complexes have also been prepared. Addition of the neutral ligand imidazole or of the anionic ligand cyanide results in formation of hexacoordinate adducts that retain thiolate ligation as determined by spectral comparison to the analogous derivatives of ferric P450cam. The stability of these complexes and their similarity to the analogous forms of P450cam illustrates the potential of the H175C/D235L CcP double mutant as a model for ferric P450 enzymes. This study marks the first time a stable cyanoferric complex of a model P450 has been made and demonstrates the importance of the environment around the primary coordination ligands in stabilizing metal-ligand ligation.     

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.     

Electron paramagnetic resonance study of ferrous cytochrome P-450scc-nitric oxide complexes: effects of cholesterol and its analogues

Electron paramagnetic resonance (EPR) spectra of nitric oxide (NO) complexes of ferrous cytochrome P-450scc were measured at 77 K for the first time without using the rapid-mixing and freeze-quenching technique. Without substrate the EPR spectra were very similar to those of cytochrome P-450cam (from Pseudomonas putida) and cytochrome P-450LM (from rat liver microsomes) with rhombic symmetry; gx = 2.071, gz = 2.001, gy = 1.962, and Az = 2.2 mT for 14NO complexes. Upon addition of substrates [such as cholesterol, 22(S)-hydroxycholesterol, 22(R)-hydroxycholesterol, 25-hydroxycholesterol, and 22-ketocholesterol], the EPR spectra exhibited many variations having rhombic symmetry in the major component and an additional minor component with less rhombic symmetry. Furthermore, addition of 20(S)-hydroxycholesterol caused a striking change in the EPR spectrum. The component with rhombic symmetry disappeared completely, and the component with less rhombic symmetry dominated (gx = 2.027, gz = 2.007, gy = 1.984, and Az = 1.76 mT for 14NO complexes). These observations suggest the existence of the following physiologically important natures: (1) the conformational flexibility of the active site of the enzyme due to the steric interaction between the substrate and the heme-bound ligand molecule and (2) the importance of the hydroxylation of the cholesterol side chain at the 20S position to proceed the side-chain cleavage reaction in cytochrome P-450scc.     

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.     

Surface enhanced resonance Raman study of phenobarbital-induced rabbit liver cytochrome P-450 LM2

Surface enhanced resonance Raman (SERR) spectroscopy has been used to study the vibrational spectra of the heme of purified rabbit liver cytochrome P-450 LM2 which was adsorbed on colloidal silver suspensions or on a silver electrode. Bases on a comparison with the resonance Raman (RR) spectra of the 'solute' species the high sensitivity of the SERR technique is demonstrated. Two different features were chosen in order to determine the structural and functional integrity of the adsorbed P-450. Both, substrate-induced spin state changes on the oxidized P-450 and the effect of the thiolate ligand on the oxidation state marker band v4 in the reduced P-450 could be observed in the SERR spectra of the adsorbed as well as in the RR spectra of the dissolved enzyme. These findings indicate that the protein structure near the substrate binding site and the coordination by thiolate are not affected by the interaction with the metal surface. Both structural elements are crucial for the function of P-450. Thus the elementary processes of the enzymatic action of P-450 can be investigated by this highly sensitive version of RR spectroscopy.     

Isolation and characterization of an altered cytochrome P-450 from a yeast mutant defective in lanosterol 14 alpha-demethylation

A cytochrome P-450 (P-450SG1) was purified from a lanosterol 14 alpha-demethylase (P-450(14DM)) defective mutant of Saccharomyces cerevisiae, strain SG1, by a method similar to that used in the purification of the wild type enzyme (Yoshida, Y., and Aoyama, Y. (1984) J. Biol. Chem. 259, 1655-1660). P-450SG1 had the same apparent Mr as and was immunochemically identical to P-450(14DM). Peptide maps of P-450SG1 made by limited proteolysis with Staphylococcus aureus V8 proteinase, chymotrypsin, or papain followed by gel electrophoresis were identical to corresponding peptide maps of P-450(14DM). However, P-450SG1 showed no lanosterol 14 alpha-demethylase activity and its mode of interaction with diniconazole [(E)-1-(2,4-dichlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-y1)-1- penten-3- o1], a specific inhibitor of P-450(14DM), was fundamentally different from that of P-450(14DM). The absorption spectrum of ferric P-450SG1 was unusual for a native low-spin cytochrome P-450 and was superimposable on that of 1-methylimidazole complex of P-450(14DM), indicating that P-450SG1 has a histidine 6th ligand trans to the thiolate 5th ligand, while the 6th ligand of other ferric low-spin cytochrome P-450s is a water molecule or a hydroxyl group of an oxyamino acid. It is concluded that P-450SG1 is an altered P-450(14DM). Difference in the primary structure between P-450SG1 and P-450(14DM) may be slight and was not detected by peptide mapping. However, the alteration caused significant change in the substrate site and heme environments of the cytochrome. P-450SG1 is the first example of a cytochrome P-450 having a histidine axial ligand trans to thiolate and of a genetically altered cytochrome P-450 isolated in a homogeneous state.     

Comparative EPR study on high-spin ferric porphine complexes and cytochrome P-450 having rhombic character

Comparative EPR studies were made on two high-spin Fe(III) porphine model systems and mammalian liver microsomal cytochromes P-450, all of which exhibit approximately the same degrees of rhombicity in their EPR spectra. Comparison of g values and linewidths as a function of temperature, and of the microwave power saturation demonstrated that EPR characteristics of P-450 are more similar to the Fe(III) porphines having the thiolate axial ligand than in the other model systems, the mixed crystals of Fe(III) porphine with the corresponding free base porphine, in which no thiolate ligand is involved.There is, however, a discrepancy between P-450 and the model thiolates with respect to the size of the zero-field parameter D. These observations indicate that P-450 heme has essential structural features in common with thiolates but the Fe-S bond of P-450 may be modified from its normal orientation in model thiolates, probably as a result of the constraints imposed by the protein stucture.     

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.     

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).     

Electron paramagnetic resonance detectable states of cytochrome P-450cam

Cytochrome P-450cam is a low-spin Fe3+hemoprotein (g = 2.45, 2.26, and 1.91) which is made 60% high spin (g = 7.85, 3.97, and 1.78) at 12 K by the addition of 1 mol of substrate per mol of enzyme. Low-temperature EPR spectra show that the low-spin fraction of substrate-bound P-450cam contains two magnetic species. The majority species has an unusual EPR spectrum (g = 2.42, 2.24, and 1.97) which connot be simulated by using the range of crystal field parameters known for other heme proteins. The minority species has the same g values as substrate-free enzyme. Both low-spin species show Curie law temperature dependence below 50 K and have similar saturation behavior. Above 50 K the g = 2.42, 2.24, and 1.97 species rapidly loses signal intensity. The distribution of low-spin species is pH dependent (apparent pKa = 6.2) with the g = 2.42, 2.24, and 1.97 magnetic species favored at high pH. The substrate binding stoichiometry and the equilibria observed in the low-spin fraction suggest that there are not multiple protein forms of cytochrome P-450cam. Putidaredoxin and other effector molecules which specifically catalyze hydroxylation convert either the high-spin or the g = 2.42, 2.24, and 1.97 low-spin species to another new magnetic species (g = 2.47, 2.26, and 1.91). This species is only seen in the presence of substrate, and its stability reflects the catalytic potency of the effector molecule. The EPR and UV-visible spectra of cytochrome P-420 depend upon the manner in which the P-420 is generated. Incubation with acetone or reaction with N-ethylmaleimide or diethyl pyrocarbonate generates P-420 with different spectral characteristics. Through identification of active-site amino acids by chemical modification and comparison with porphyrin model complexes, the range of ligands likely to participate in each of the EPR detectable species is assigned. Mechanisms of interconversion of these species and their bearing on catalysis are discussed.