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We have analyzed the buried water molecules and internal cavities in a set of 75 high-resolution, nonhomologous, monomeric protein structures. The number of hydrogen bonds formed between each water molecule and the protein varies from 0 to 4, with 3 being most common. Nearly half of the water molecules are found in pairs or larger clusters. Approximately 90% are shown to be associated with large cavities within the protein, as determined by a novel program, PRO_ACT. The total volume of a protein's large cavities is proportional to its molecular weight and is not dependent on structural class. The largest cavities in proteins are generally elongated rather than globular. There are many more empty cavities than hydrated cavities. The likelihood of a cavity being occupied by a water molecule increases with cavity size and the number of available hydrogen bond partners, with each additional partner typically stabilizing the occupied state by 0.6 kcal/mol.
P450 hemeproteins comprise a large gene superfamily that catalyzes monooxygenase reactions in the presence of a redox partner. Because the mammalian members are, without exception, membrane-bound proteins, they have resisted structure-function analysis by means of X-ray crystallographic methods. Among P450-catalyzed reactions, the aromatase reaction that catalyzes the conversion of C19 steroids to estrogens is one of the most complex and least understood. Thus, to better understand the reaction mechanism, we have constructed a three-dimensional model of P450arom not only to examine the active site and those residues potentially involved in catalysis, but to study other important structural features such as substrate recognition and redox-partner binding, which require examination of the entire molecule (excepting the putative membrane-spanning region). This model of P450arom was built based on a "core structure" identified from the structures of the soluble, bacterial P450s (P450cam, P450terp, and P450BM-P) rather than by molecular replacement, after which the less conserved elements and loops were added in a rational fashion. Minimization and dynamic simulations were used to optimize the model and the reasonableness of the structure was evaluated. From this model we have postulated a membrane-associated hydrophobic region of aliphatic and aromatic residues involved in substrate recognition, a redox-partner binding region that may be unique compared to other P450s, as well as residues involved in active site orientation of substrates and an inhibitor of P450arom, namely vorozole. We also have proposed a scheme for the reaction mechanism in which a "threonine switch" determines whether oxygen insertion into the substrate molecule involves an oxygen radical or a peroxide intermediate.
Methane monooxygenase (MMO), found in aerobic methanotrophic bacteria, catalyzes the O2-dependent conversion of methane to methanol. The soluble form of the enzyme (sMMO) consists of three components: a reductase, a regulatory "B" component (MMOB), and a hydroxylase component (MMOH), which contains a hydroxo-bridged dinuclear iron cluster. Two genera of methanotrophs, termed Type X and Type II, which differ markedly in cellular and metabolic characteristics, are known to produce the sMMO. The structure of MMOH from the Type X methanotroph Methylococcus capsulatus Bath (MMO Bath) has been reported recently. Two different structures were found for the essential diiron cluster, depending upon the temperature at which the diffraction data were collected. In order to extend the structural studies to the Type II methanotrophs and to determine whether one of the two known MMOH structures is generally applicable to the MMOH family, we have determined the crystal structure of the MMOH from Type II Methylosinus trichosporium OB3b (MMO OB3b) in two crystal forms to 2.0 A resolution, respectively, both determined at 18 degrees C. The crystal forms differ in that MMOB was present during crystallization of the second form. Both crystal forms, however, yielded very similar results for the structure of the MMOH. Most of the major structural features of the MMOH Bath were also maintained with high fidelity. The two irons of the active site cluster of MMOH OB3b are bridged by two OH (or one OH and one H2O), as well as both carboxylate oxygens of Glu alpha 144. This bis-mu-hydroxo-bridged "diamond core" structure, with a short Fe-Fe distance of 2.99 A, is unique for the resting state of proteins containing analogous diiron clusters, and is very similar to the structure reported for the cluster from flash frozen (-160 degrees C) crystals of MMOH Bath, suggesting a common active site structure for the soluble MMOHs. The high-resolution structure of MMOH OB3b indicates 26 consecutive amino acid sequence differences in the beta chain when compared to the previously reported sequence inferred from the cloned gene. Fifteen additional sequence differences distributed randomly over the three chains were also observed, including D alpha 209E, a ligand of one of the irons.
The kinetics and equilibrium of carbon monoxide binding and the reaction of autodecomposition of oxycomplexes of two bacterial P450s--P450BM-3 and P450terp--have been studied and compared with the corresponding properties of P450cam and some of the microsomal P450s. The second-order reaction rate constants for the reaction of P450terp and P450BM-3 with carbon monoxide in the absence of substrate at 5 degrees C were 3.6 x 10(6) and 1.6 x 10(6) M-1 s-1, respectively. The presence of the physiological substrate markedly influenced the rate of carbon monoxide binding with P450terp, decreasing the rate constant by approximately 100-fold, and did not have a significant effect on carbon monoxide binding with P450BM-3. The reaction of carbon monoxide with both cytochromes was monophasic in the absence or in the presence of substrate. The carbon monoxide binding enthalpy change was very small for P450BM-3 (-0.2 kcal mol-1) and more negative for P450terp (-3.2 kcal mol-1). The rate constants of decomposition of oxy-P450terp and oxy-P450BM-P (heme domain of P450BM-3) at 5 degrees C were 7.1 x 10(-4) and 2.5 x 10(-2) s-1, respectively. Raising the temperature to 20 degrees C resulted in 24- and 9-fold increase of decomposition of oxy-complexes of P450terp and P450BM-P, respectively. The kinetic properties of the binding reaction of diatomic gases to P450cam, P450terp, and P450BM-3 are consistent with the structures of their active sites.
Recent structural studies indicate that the substrate- and O2-binding distal pocket of the P450 enzymes are not identical. Thus, P450terp (CYP108) from the alpha-terpineol-metabolizing Pseudomonad differs from P450cam (CYP-101) (C. A. Hasemann et al., J. Mol. Biol. 236, 1169, 1994). In contrast, the distal pockets of P450terp and P450BMP (CYP102 heme domain; Bacillus megaterium) are more closely similar, including novel hydrogen-bonding interactions between the distal H2O ligand and the I helix (C. A. Hasemann et al., Structure, 3, 41-62, 1995). To evaluate the significance of these differences, we have compared solution magnetic circular dichroism (MCD) spectra of P450terp with spectra of other P450 enzymes (e.g., P450cam, P450BMP, P450BM-3holo, and P450BM1), as well as with spectra of chloroperoxidase and NO synthase. Spectra of native P450terp are more similar to those of P450BMP and those of mammalian P450LM-2 than to those of P450cam. Upon substrate-binding, the MCD spectra of ferric P450terp and all other thiolate-ligated heme systems examined to date display a strong Soret band that is distinctly unique relative to the typical Soret MCD pattern(s) of catalases or other 5-coordinate ferric heme systems. This intense negative MCD feature thus appears diagnostic for cysteinate-linked ferric hemes. In the case of ferrous P450s, the intensity of the Soret-region MCD trough varies between substrate-bound and substrate-free enzymes (despite the fact that the substrate is NOT in direct contact with the heme moiety). A novel finding of particular interest is the clear spectral shifts of the Soret MCD band between the substrate-bound and substrate-free forms of ferrous-CO-P450terp. No such observation has been made previously. Furthermore, the band positions for BOTH types of P450terp are red-shifted from known bands of ferrous-CO-P50cam. These data thus indicate a surprising sensitivity of MCD spectra to active-site polarity and to H2O occupancy, concurring with reports of distal pocket effects on CO-binding rates and equilibrium constants. Comparative analysis of the spectral properties of P450terp with MCD spectra of other P450 enzymes, as well as with chloroperoxidase and NO synthase, demonstrates both the expected similarities and the significant differences that reflect active-site structural features. The detailed spectral analysis of P450terp relative to other P450 enzymes presented herein includes the first observation of a substrate-induced spectral shift for a ferrous-CO-P450. Furthermore, testable structural predictions for P450-BM-1 and for the novel NO synthase enzyme (neither of which has been crystallized to date) are made herein. This work thus provides insights into structurally defined P450s and may also lead to understanding of other P450 enzymes.
Eighteen linear antigenically active sites were revealed in cytochrome P450 from Pseudomonas putida (P450cam) by hexapeptide scanning. These sites occupy about 31% of the protein sequence. Hexapeptide epitope sequences of P450cam are not found in other cytochromes P450. However, several cytochromes P450 contain shorter fragments of P450cam epitope sequences which may cause weak immune cross-reactions. P450cam antigenic determinants are located generally at the boundaries of secondary structure elements. Mapping of P450cam antigenic determinants on the three-dimensional structure of this protein reveals 14 highly water-accessible antigenic sites and only 1 site (No. 322-327, QMLSGL) which is inaccessible to water. Several functionally important sites and amino acid residues of P450cam are localized within revealed linear epitopes or very close to them. These sites include substrate-binding regions, residues responsible for the putidaredoxin interaction (Arg72, Arg112, Lys314, and Arg364), heme binding (Gln108, Arg112, Asp297, Arg299, and Cys357), and proton translocation (Lys178, Arg186, and Glu366).
The oxidation by cytochrome P450cam (CYP101) of ethylbenzene and a series of substrates derived from it by addition of one, two, three, or four carbon atoms has been examined. For each of the 18 substrates, the shift in spin state due to substrate binding, the extent of coupled turnover to give organic products and uncoupled turnover to give H2O2 and H2O, and the identities of the organic products have been determined. The same studies have been carried out with the T185L and T185F mutants of P450cam in which the active site volume is decreased. The results show that no detectable correlation exists between the observed spin state change and any other parameter studied. For substrates of equal size, coupled and uncoupled turnover vary widely but both are maximized when the phenyl ring bears one large alkyl substituent or a methyl ortho to the largest alkyl substituent. The presence of substituents in addition to these decreases activity. In the absence of other changes, coupled turnover is correlated with the size of the largest substituent, but no such correlation exists for uncoupled turnover. Decreasing the size of the active site cavity by a T185L mutation generally increases coupled turnover without altering the dependence on the alkyl group size. A T185F mutation causes too great an active site perturbation for structure-activity studies. Substrate oxidation occurs preferentially at 2 degrees or 3 degrees C-H bonds of the largest substituent or on the benzylic methyl ortho to it. Aromatic hydroxylation only competes with the oxidation of nonbenzylic 1 degree C-H bonds. The extent of coupled turnover is a function of substrate shape, substrate size, and cavity size, but still elusive parameters control the extent of uncoupled turnover.
In the present study, two P450s (P4502B4 and P450cam) have been examined with regard to their interactions with their substrates and products utilizing the characteristic spectral perturbations as criteria for their binding. The results indicate that although there are differences between the two P450s (E) in regard to their precise interactions with their substrates (S) and products (P), the spectral titration data were consistent with the two-site model--E + S<-->ES (K1), E + P<-->EP (K2); EP + S<-->ESP (K3); ES + P<-->ESP (K4) in which S and P bind to E forming ESP. The data were inconsistent with the two-site model in which S and P compete for the same site. As required by the two-site model, the relationship K2K3 = K1K4 was maintained with both P450s at all product concentrations tested, although K3 and K4 decreased considerably when product concentration was increased. The relationship K3 >> K4 was also maintained, indicating that with both enzymes' ESP is formed predominantly by binding of S to EP rather than binding of P to ES, and that ESP dissociates predominantly to ES and P rather than EP and S. In other words, binding of S to EP facilitates the dissociation of P. This indicates that the relative parameter values are compatible for ESP to have functional significance. The possible role of ESP in controlling catalytic rate and catalytic efficiency is discussed.
A real-time optical biosensor study on the interactions between putidaredoxin reductase (PdR), putidaredoxin (Pd), and cytochrome P450cam (P450cam) within the P450cam system was conducted. The binary Pd/P450cam and Pd/PdR complexes were revealed and kinetically characterized. The dominant role of electrostatic interactions in formation of productive electron transfer complexes was demonstrated. It was found that Pd/P450cam complex formation and decay obeys biphasic kinetics in contrast to the monophasic one for complexes formed by other redox partners within the system. Evidence for PdR/P450cam complex formation was obtained. It was found that, in contrast to Pd, which binds only to its redox partners, PdR and P450cam were able to form PdR/PdR and P450cam/P450cam complexes. A ternary PdR/Pd/P450cam complex was also registered. Its lifetime was sufficient to permit up to 60 turnovers to occur. The binding of Pd to P450cam and to PdR within the ternary complex occurred at distinct sites, with Pd serving as a bridge between the two proteins.
Protein continuous B-epitopes can be revealed using short synthetic peptides that overlap a known protein sequence. Since the whole protein surface is considered to possess antigenic properties, a question that arises is whether a set of linear B-epitopes determined by peptide scanning correlates with a protein spatial structure. We have chosen cytochrome P450cam (CYP101) of Pseudomonas putida, with known 3D structure, as a template. Sera of two rabbits and antibody egg yolk preparations from three chickens were produced against the P450cam molecule. These polyclonals were analyzed separately in ELISA with 409 overlapping P450cam hexapeptides. The whole set of continuous antigenic sites of P450cam covered about 45% of the P450cam sequence. However, immunodominant sites (those revealed with more than 50% antibody preparations), the so-called "antigenic core," represent only 9% of the protein sequence. While the amount of water-accessible residues in the total antigenic map (42%) was close to that in the whole native P450cam molecule (39%), the amount of water-accessible residues in the antigenic core was significantly higher (64%). These results led to the conclusion that antigenic core epitopes can be associated to the molecular surface, whereas epitopes with low detection frequency may partly correspond to unfolded regions of the protein molecule.
Three factors are of primary importance with respect to designing efficient P450 biocatalysts. (1) The substrate must be oxidized at a significant rate. (2) The regioselectivity must heavily favor the desired product. (3) The enzyme must use the majority of the reducing equivalents from NADH or NADPH to produce product. The reaction we chose to study was oxidation of 2-ethylhexanol to 2-ethylhexanoic acid by P450cam. We examined four active site mutations: F87W, Y96W, T185F, and L244A. The mutations were chosen to improve 2-ethyhexanoic acid production by decreasing active site volume, increasing active site hydrophobicity, and improving stereoselectivity. The F87W and Y96W mutations improved regioselectivity, giving almost exclusively the desired product. The T185F mutation improved coupling of NADH to product formation. The L244A mutation altered the stereoselectivity of 2-ethylhexanoic acid production. These results indicate that active site mutations of P450cam can alter catalysis of 2-ethylhexanol.
Cytochrome P450CAM from Pseudomonas putida G786 oxidized chlorinated ethanes and 1,2-dichloropropane. The rate of NADH oxidation decreased with decreasing chlorine substitution. The single detectable oxidation products of 1,1,1-trichloroethane and 1,2-dichloropropane were 1,1,1-trichloroethanol and chloroacetone, respectively. Organic product formation was largely uncoupled from NADH oxidation.
During the reaction of m-chloroperbenzoate with low spin ferric form of cytochrome P450cam, the formation of at least four transient intermediates was detected by employing rapid scan absorption spectrometry. Among them, the first one which appeared within 10 msec after the start of reaction gave an absorption spectrum indistinguishable from that of compound I of chloroperoxidase, another thiolate-heme protein. The intermediate exhibited the major absorption maxima at 367 and 694 nm, while chloroperoxidase compound I is known to have the maxima at 367 and 688 nm. The shapes of the bands at 367 nm were very similar to each other. Based on these spectral similarities, we suggest that the intermediate found in this study is the compound I of cytochrome P450cam.
Replacement of Thr-252 in the active center of cytochrome P450cam with a non-hydroxy amino acid residue such as Ala and Val by conventional site-directed mutagenesis converted this monooxygenase to an NADH oxidase (Imai, M. et al. Proc. Natl. Sci. U. S. A. 86, 7823-7827, 1989). In this study, a mutant enzyme with a methoxy group in place of the hydroxy group of Thr-252 (OMe-mutant) was synthesized by the method of unnatural amino acid mutagenesis (Noren, C. J. et al., Science 244, 182-188, 1989). Unlike other site-directed mutants without a hydroxy group at the position, the OMe-mutant retained a considerably high monooxygenase activity, yielding a stoichiometric amount of 5-exo-hydroxycamphor to that of the oxygen consumed. Thus a free hydroxy group at this position is not an indispensable requisite for the monooxygenase to cleave the O-O bond of molecular O2 as previously proposed.
Sedimentation experiments on cytochrome P450cam (CYP101) has been performed to compare the molecular mass of the protein in the oxidized state and as carbon monoxide complex. The oxidized protein in the absence of beta-mercaptoethanol is a dimer with a molecular mass of 92 kDa. Addition of mercaptoethanol avoids completely the dimerization. Dithionite reduced P450cam in the presence of carbon monoxide has been found to be a monomeric protein.
We demonstrate a very fast heterogeneous redox reaction of substrate-free cytochrome P450(cam) on a glassy carbon electrode modified with sodium montmorillonite. The linear relationship of the peak current in the cyclic voltammogram with the scan rate indicates a reversible one-electron transfer surface process. The electron transfer rate is in the range from 5 to 152 s(-1) with scan rates from 0.4 to 12 V/s, respectively. These values are comparable to rates reported for the natural electron transfer from putidaredoxin to P450(cam). The formal potential of adsorbed P450(cam) is -139 mV (vs NHE) and therefore positively shifted by 164 mV compared to the potential of substrate-free P450(cam) in solution. UV-VIS and FTIR spectra do not indicate an influence of the clay colloidal particles on the heme and the secondary structure of P450(cam) in solution. However, P450(cam) adsorbed on the surface of the clay-modified electrode may undergo partial dehydration resulting in the shift of the formal potential.