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.     

Flash photolysis studies on the CO complexes of ferrous cytochrome P-450scc and cytochrome P-45011 beta. Effects of steroid binding on the photochemical and ligand binding properties

Upon irradiation by a light flash (100-J), the carbon monoxide complex of cytochrome P-450scc was fully photodissociated in both the presence and absence of cholesterol, while less than 20% of the CO complex was photodissociable with those of deoxycorticosterone-bound and -free forms of cytochrome P-45011 beta. When the quantum yield of the reaction was measured for each photodissociable portion, the values were 0.5 and 1.0 for the substrate-free and -bound forms of cytochrome P-450scc, and 0.03 and 0.8 for the substrate-free and -bound forms of cytochrome P-45011 beta, respectively. Thus, CO complexes of these enzymes become more photosensitive upon binding with the specific substrates. Steroid binding also affected kinetic constants of reactions between the ferrous enzymes and CO. The rate constants for the CO recombination at 15 degrees C were 2.7 X 10(6) and 2.3 X 10(5) M-1 s-1 for the substrate-free and -bound forms of cytochrome P-450scc, and were 7.0 X 10(5) and 5.4 X 10(3) M-1 s-1 for the substrate-free and -bound forms of cytochrome P-45011 beta, respectively. The rate constants for the CO dissociation also decreased upon the steroid bindings. The products of the enzyme reactions, pregnenolone and corticosterone, had similar effects on the kinetic constants. From these findings, we postulate that the binding of a steroid to the substrate site of each enzyme alters the bonding character of CO with the heme-iron, thereby affecting both photochemical and kinetic properties of the CO complex. The nature of the photoindissociable portion of the CO complex of cytochrome P-45011 beta is also discussed.     

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.     

Proton nmr spectroscopy of high-spin ferrous cytochrome P-450 models

Alkyl mercaptide complexes of both synthetic and natural-derivative iron(II) porphyrins have been characterized in DMSO solution by proton nmr spectroscopy. A single mercaptide ligand binds to form a high-spin iron(II) complex as determined by solution magnetic measurements and the nmr isotropic shift pattern. Ligand exchange is slow on the nmr time scale unlike corresponding 2-methyl imidazole exchange rates which are very rapid. Further comparison of mercaptide and 2-methyl imidazole adducts reveals a downfield bias in isotropic shift values for the mercaptide species, which may be explained by different signs in the dipolar shift term for the two complexes. This apparent magnetic anisotropy of the mercaptide complex is in the same direction, although smaller, than that observed for bacterial cytochrome P-450. Isotropic shift values of at least 250 ppm for methylene resonances of the coordinated mercaptide support a very efficient unpaired spin delocalization for this axial ligand.     

Induction of cytochrome P-450 in rat liver by the antibiotic troleandomycin: partial purificaton and properties of cytochrome P-450-troleandomycin metabolite complexes

Liver cytochrome P-450 from rats treated intraperitoneally with troleandomycin (TAO) were solubilized and partially purified using DE 52 anion exchange chromatography. The major TAO-induced cytochrome P-450 form appears in fraction A which is not bound on the DE 52 column. It is different from the major form induced in rats by phenobarbital or 3-methylcholanthrene in terms of absolute visible spectroscopy, gel electrophoresis (M 45000) and reactions with antibodies. This TAO-induced form mainly exists $\text{in vivo}$ as an iron-TAO metabolite complex and exhibits a characteristic Soret peak at 456 nm. Reconstitution experiments using this partially purified form, after dissociation of its iron-metabolite bond by ferricyanide treatment, underline its particular ability to demethylate TAO itself. TAO also leads to an important induction of other cytochromes P-450 that are present in fraction B (retained on DE 52 column) like the major phenobarbital-induced form, but are immunologically distinct from it.     

Ligand and halide binding properties of chloroperoxidase: peroxidase-type active site heme environment with cytochrome P-450 type endogenous axial ligand and spectroscopic properties

Equilibrium binding studies of exogenous ligands and halides to the active site heme iron of chloroperoxidase have been carried out from pH 2 to 7. Over twenty ligands have been studied including C, N, O, P, and S donors and the four halides. As judged from changes in the optical absorption spectra, direct binding of the ligands to the heme iron of ferric or ferrous chloroperoxidase occurs in all cases; this has been ascertained for the ferric enzyme in several cases through competition experiments with cyanide. All of the ligands except for the halides, nitrate, and acetate form exclusively low-spin complexes in analogy to results obtained with the spectroscopically related protein, cytochrome P-450-CAM [Sono, M., & Dawson, J.H. (1982) J. Biol. Chem. 257, 5496-5502]. The titration results show that, for the ferric enzyme, (i) weakly acidic ligands (pKa greater than 3) bind to the enzyme in their neutral (protonated) form, followed by deprotonation upon ligation to the heme iron. In contrast, (ii) strongly acidic ligands (pKa less than 0) including SCN-, NO3-, and the halides except for F- likely bind in their anionic (deprotonated) form to the acid form of the enzyme: a single ionizable group on the protein with a pKa less than 2 is involved in this binding. For the ferrous enzyme, (iii) a single ionizable group with the pKa value of 5.5 affects ligand binding. These results reveal that chloroperoxidase, in spite of the previously established close spectroscopic and heme iron coordination structure similarities to the P-450 enzymes, clearly belongs to the hydroperoxidases in terms of its ligand binding properties and active site heme environment. Magnetic circular dichroism studies indicate that the alkaline form (pH 9.5) of ferric chloroperoxidase has an RS-ferric heme-N donor ligand coordination structure with the N donor likely derived from histidine imidazole.     

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.     

Direct formation of complexes between cytochrome P-450 and nitrosoarenes

The mechanism of the formation of the complexes between various nitrosobenzenes and cytochrome P-450 has been investigated. We have observed the formation of these complexes by a new and, as yet, undescribed route. Nitrosobenzene (NOB) itself reacts with cytochrome P-450 in the iron(III) state, in the absence of any exogenous reducing agent, to produce the iron(II)-NOB complex. Apparently, NOB is a ligand that is capable of causing the spontaneous autoreduction of the iron. The reduction of the iron may occur via ligand-induced oxidation of the axially bound thiolate of cytochrome P-450.     

Cleavage of DNA by model complexes of cytochrome P-450

Thiolate-hemin complexes as chemical models for cytochrome P-450 have been shown to cause cleavage of DNA. The cleavage of DNA to open-circular and linear forms depended on the structure of thiol ligand and the thiol ligand:hemin ratio at pH 7.8. Complete cleavage of DNA was observed by complexes containing thioglycolate ethylester and mercaptoethanol at 400-600 moles excess of thiol ligand to hemin, those containing cysteine, cysteine methylester and cysteine ethylester at 50-200 moles excess, and those containing mercaptopropionylglycine, glutathione, glutathione dimethylester, penta- and nonapeptides at 5-100 moles excess. Inhibition experiments suggested the involvement of active oxygen species in the cleavage of DNA.     

Unusual spectroscopic and ligand binding properties of the cytochrome P450-flavodoxin fusion enzyme XplA

The Rhodococcus rhodochrous strain 11Y XplA enzyme is an unusual cytochrome P450-flavodoxin fusion enzyme that catalyzes reductive denitration of the explosive hexahydro-1,3,5-trinitro-1,3,5-triazene (RDX). We show by light scattering that XplA is a monomeric enzyme. XplA has high affinity for imidazole (K(d) = 1.6 μM), explaining previous reports of a red-shifted XplA Soret band in pure enzyme. The true Soret maximum of XplA is at 417 nm. Similarly, unusually weak XplA flavodoxin FMN binding (K(d) = 1.09 μM) necessitates its purification in the presence of the cofactor to produce hallmark flavin contributions absent in previously reported spectra. Structural and ligand-binding data reveal a constricted active site able to accommodate RDX and small inhibitory ligands (e.g. 4-phenylimidazole and morpholine) while discriminating against larger azole drugs. The crystal structure also identifies a high affinity imidazole binding site, consistent with its low K(d), and shows active site penetration by PEG, perhaps indicative of an evolutionary lipid-metabolizing function for XplA. EPR studies indicate heterogeneity in binding mode for RDX and other ligands. The substrate analog trinitrobenzene does not induce a substrate-like type I optical shift but creates a unique low spin EPR spectrum due to influence on structure around the distal water heme ligand. The substrate-free heme iron potential (-268 mV versus NHE) is positive for a low spin P450, and the elevated potential of the FMN semiquinone/hydroquinone couple (-172 mV) is also an adaptation that may reflect (along with the absence of a key Thr/Ser residue conserved in oxygen-activating P450s) the evolution of XplA as a specialized RDX reductase catalyst.     

On the mechanism of action of cytochrome P-450. Oxidation and reduction of the ferrous dioxygen complex of liver microsomal cytochrome P-450 by cytochrome b5

The effects of cytochrome b5 on the decay of the ferrous dioxygen complexes of P-450LM2 and P-450LM4 from rabbit liver microsomes were studied by stopped-flow spectrophotometry. The P-450 (FeIIO2) complexes accept an electron from reduced cytochrome b5 and, in a reaction not previously described, donate an electron to oxidized cytochrome b5 to give ferric P-450. A comparison with the electron-transferring properties of ferrous P-450 under anaerobic conditions allowed determination of the limiting steps of the two reactions involving the oxygenated complex. The rate of decay of the dioxygen complex was increased in all cases with b5 present; however, with oxidized b5 a large increase in the rate was observed with P-450 isozyme 4 but not with isozyme 2, whereas the opposite situation was found when reduced b5 was used. The reactions between b5 and ferrous dioxygen P-450 were not at thermodynamic equilibrium under the conditions employed. From the results obtained, a model is proposed in which the ferrous dioxygen complex decomposes rapidly into another species differing from ferric P-450 in its spectral properties and from the starting complex in its electron-transferring properties. A scheme is presented to indicate how competition among spontaneous decay, cytochrome b5 oxidation, and cytochrome b5 reduction by the ferrous O2 complex may influence substrate hydroxylation.     

Insect cytochrome P-450

Summary Two approaches may be used to study the function of cytochrome P-450 in insects: (a) an evaluation of the spectral and catalytic properties of the hemoprotein while associated with microsomal membranes; (b) the solubilization, resolution and purification of the microsomal mixed-function oxidase system. The first approach has provided some understanding of the biochemical factors involved in the metabolism of a variety of compounds, including pesticides, drugs, hormones and many other xenobiotics. However, solubilization of the monooxygenase system allows the study of each of its components individually, providing a better insight on the sequence of events leading to the hydroxylation of a substrate, the type of intermediates formed, and the rate-limiting step(s). This report discusses studies carried out with the monooxygenase system associated with microsomal membranes, as well as procedures to solubilize and partially purify its components from housefly microsomes. The latter involves solubilization with either Triton X-100 or sodium cholate, followed by either ammonium sulfate fractionation, Sephadex G-200, DEAE-Sephadex A-50 column chromatography or by-amino-n-octyl-Sepharose 4B affinity chromatography. These procedures have shown that two cytochrome P-450 species (P-450 and P-450_I) are present in microsomes isolated from a resistant housefly strain. Induction with either naphthalene or phenobarbital appears to increase cytochrome P-450_I preferentially.An invited article.     

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.     

Catalytic properties of cytochrome P-450 from human liver

A partially purified cytochrome P-450 fraction was prepared from the microsomal fraction of human liver. When combined with NADPH, a synthetic phospholipid and NADPH-cytochrome P-450 reductase from rat liver, the cytochrome P-450 fraction from human liver was able to catalyze the following hydroxylations: 11- and 12-hydroxylation of laurate, 12α- and 26-hydroxylation of 5β-cholestane-3α,7α-diol, 25-hydroxylation of 5β-cholestane-3α,7α,12α-triol, and 6β-hydroxylation of androstenedione and progesterone. It was shown that the rate of 11- and 12-hydroxylation of laurate was linear with increasing amounts of cytochrome P-450 and with time in the presence of excess NADPH-cytochrome P-450 reductase and the phospholipid. In the presence of a fixed amount of cytochrome P-450 and the phospholipid, the rate of 11- and 12-hydroxylation increased with increasing concentrations of NADPH-cytochrome P-450 reductase up to a certain level and then remained constant. The requirement of the phospholipid could be increased markedly by centrifugation of the cytochrome P-450 fraction at 100,000g just prior to incubation. It is concluded that cytochrome P-450 from human liver is similar to previously studied cytochrome P-450 from rat liver with respect to catalytic properties and mechanism of reaction.     

Cobalt-substituted cytochrome P-450cam

Reconstitution of the apo-cytochrome with cobalt protoporphyrin provides a faithful P-450cam analogue as characterized by optical, ligand-binding, and enzymatic parameters. The thiol and cyanide complexes exhibit Soret "hyper" spectra, not previously observed in cobalt porphyrins. Substrate-induced spectral changes and limited stereospecific hydroxylation activity are retained in the cobalt P-450cam. The EPR (electron paramagnetic resonance) spectra of the reduced cobaltous protein indicate clearly an endogenous axial ligand other than a nitrogenous base and support an assignment of thiolate coordination. A thiolate ligand is also indicated by EPR measurements in the oxygenated cobaltous analogue. By analogy, these studies suggest that the native ferrous and oxygenated P-450cam states retain a thiolate axial ligand.