Spring-loading the active site of cytochrome P450cam

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

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

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

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.     

Hydration energy landscape of the active site cavity in cytochrome P450cam

Hydration of protein cavities influences protein stability, dynamics, and function. Protein active sites usually contain water molecules that, upon ligand binding, are either displaced into bulk solvent or retained to mediate protein–ligand interactions. The contribution of water molecules to ligand binding must be accounted for to compute accurate values of binding affinities. This requires estimation of the extent of hydration of the binding site. However, it is often difficult to identify the water molecules involved in the binding process when ligands bind on the surface of a protein. Cytochrome P450cam is, therefore, an ideal model system because its substrate binds in a buried active site, displacing partially disordered solvent, and the protein is well characterized experimentally. We calculated the free energy differences for having five to eight water molecules in the active site cavity of the unliganded enzyme from molecular dynamics simulations by thermodynamic integration employing a three-stage perturbation scheme. The computed free energy differences between the hydration states are small (within 12 kJ mol⁻¹) but distinct. Consistent with the crystallographic determination and studies employing hydrostatic pressure, we calculated that, although ten water molecules could in principle occupy the volume of the active site, occupation by five to six water molecules is thermodynamically most favorable. Proteins 32:381–396, 1998. © 1998 Wiley-Liss, Inc.     

Binding sites of pyridine in cytochrome P-450cam

Careful titration of oxidized cytochrome P-450cam from Pseudomonas putida with pyridine revealed deviations of the Eadie plot from linearity in the substrate-bound as well as in the substrate-free protein. A binding model which assumes two binding sites for pyridine--the iron and the camphor binding site--is able to describe completely the nonlinear Eadie plot.     

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.     

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.     

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

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

Conformational relaxation in hemoproteins: the cytochrome P-450cam case

Photodissociation of (CO)P-450(cam)(substrate) complexes was found to trigger a conformational relaxation process that interferes with ligand rebinding at temperatures as low as 140 K even though the protein conformational substates (CS(1)) remain frozen. To analyze the rebinding and relaxation kinetics, we developed a model that takes the distribution of relaxation rates explicitly into account and in which rebinding and relaxation rates are connected by a linear free energy relation. In all complexes heme relaxation occurs first and is probably faster than 100 ns even at 77 K. This is the only process found in substrate-free P-450(cam). Above 140 K and in the presence of a substrate, this initial, fast rebinding state (P) progressively relaxes to another state (P degrees ) in which rebinding is slower. The relaxation rate is independent of solvent rigidity and is governed by the protein's internal dynamics. Rebinding enthalpies in P and P degrees as well as the enthalpy shift brought about by relaxation correlate with the substrate propensity to block access to the iron site. In P degrees the barrier is higher because the substrate is closer to the heme normal and exerts more steric repulsion for CO binding. The relaxation process implies the return of substrate and heme to their ligand-free positions in which access to the heme is reduced.     

Interaction of a spin-labelled cholesterol derivative with the cytochrome P-450scc active site

The cholesterol analogue 25-doxyl-27-nor-cholesterol (CNO), was found to be a substrate for cytochrome P-450scc. Upon incubation with the cytochrome P-450scc electron transfer system, CNO is transformed to pregnenolone (Km = 33 microM, Vmax = 0.32 min-1). The pregnenolone formation from endogenous cholesterol is strongly inhibited by CNO (50% at 5 microM). It binds tightly to cytochrome P-450scc as evidenced by a reversed type I spectral absorbance change (Kd = 5.9 microM) which is paralleled by a greater hyperfine splitting of the room-temperature CNO ESR spectrum due to an enhanced probe immobilization (Kd = 1.9 microM). This finding is in accord with a rotational correlation time of about 10(-7) s, which is close to the tumbling rate of the protein. At 110 K the CNO-bound cytochrome P-450scc displays the ESR g-values gx = 2.404/2.456, gy = 2.245 and gz = 1.916; these are different from those of cholesterol-liganded cytochrome P-450scc and may thus serve as a marker for cytochrome P-450scc. Our data indicate that the stereospecificity of the cytochrome P-450scc side-chain-cleaving activity is not dependent on the nature of the cholesterol side-chain termination (C25 to C27). The substrate binding site is however rather sensitive to a modification of the side chain. The doxyl ring confers a stronger affinity of the substrate to the enzyme. Upon binding it becomes embedded in the protein matrix, and we estimate that its final position is 0.6-1.0 nm from the heme moiety.     

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.     

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.     

Proton activities for three states of cytochrome P-450cam.

Changes in proton concentration during the binding of dioxygen, carbon monoxide, and for the exchange of dioxygen by carbon monoxide, at ferrous-cytochrome P-450cam were measured by direct titration. Insufficient proton release was observed to support protonation-deprotonation of an axial cysteinyl sulfur donor as a mechanism for generation of hyper spectra in only the carbonylated ferrous state. Measurement of the $\text{P}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ value for CO binding as a function of pH (the carbon monoxide Bohr effect) confirms the direct titration data.     

Synthetic active site analogues of heme-thiolate proteins. Characterization and identification of intermediates of the catalytic cycles of cytochrome P450cam and chloroperoxidase

In view of recent results from different sources, the reaction mechanisms of two heme-thiolate proteins, cytochrome P450cam and chloroperoxidase (CPO), are discussed. In this context a mechanism of CPO is proposed which includes H2O2 cleavage, subsequent formation of compound I and the identification of two elusive intermediates. The HOCl adduct of the iron(III)porpyhrin is the catalytically competent Cl+ donor chlorinating activated C-H bonds of substrates bound to the enzyme. Pulse-EPR characterization of an enzyme model of the resting state of P450cam suggests a role of the electric field of the protein for stabilizing the low-spin state of the cofactor of the enzyme. It is further suggested that the same effect of the protein may trigger the reactivity of compound I such that both concerted and two-step reactions are feasible within the concept of a Two-State-Reactivity. This review emphasizes the value of synthetic enzyme models complementing investigations of the native proteins.     

Purification and characterization of diabetes-inducible cytochrome P-450

A diabetes-inducible form of cytochrome P-450, termed P-450DM, was purified to electrophoretical homogeneity (MW 51,000) by high-performance liquid chromatography from liver microsomes of diabetic rats induced with streptozotocin. The CO-reduced absorption maximum of P-450DM was at 452 nm and the oxidized heme iron appeared to be predominately in the high-spin state as deduced from the Soret maximum at 395 nm. P-450DM was active in aniline hydroxylation and N-nitrosodimethylamine demethylation. The dealkylation activity toward 7-ethoxycoumarin by P-450DM was much enhanced by the addition of cytochrome b5.     

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.