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PubMed1997

Identification of a functional water channel in cytochrome P450 enzymes.

Cytochrome P450 enzymes are monooxygenases that contain a functional heme b group linked to a conserved cysteine with a thiolate bond. In the native state, the central iron atom is hexacoordinated with a covalently bound water molecule. The exclusion of solvent molecules from the active site is essential for efficient enzymatic function. Upon substrate binding, water has to be displaced from the active site to prevent electron uncoupling that results in hydrogen peroxide or water. In contrast to typical hemoproteins, the protein surface is not directly accessible from the heme of cytochromes P450. We postulate a two-state model in which a conserved arginine, stabilizing the heme propionate in all known cytochrome P450 crystal structures, changes from the initial, stable side-chain conformation to another rotamer (metastable). In this new state, a functional water channel (aqueduct) is formed from the active site to a water cluster located on the thiolate side of the heme, close to the protein surface. This water cluster communicates with the surface in the closed state and is partly replaced by the flipping arginine side chain in the open state, allowing water molecules to exit to the surface or to reaccess the active site. This two-state model suggests the presence of an exit pathway for water between the active site and the protein surface.

Amino Acid Sequence↗

PubMed1999

Optical detection of cytochrome P450 by sensitizer-linked substrates.

The ability to detect, characterize, and manipulate specific biomolecules in complex media is critical for understanding metabolic processes. Particularly important targets are oxygenases (cytochromes P450) involved in drug metabolism and many disease states, including liver and kidney dysfunction, neurological disorders, and cancer. We have found that Ru photosensitizers linked to P450 substrates specifically recognize submicromolar cytochrome P450(cam) in the presence of other heme proteins. In the P450:Ru-substrate conjugates, energy transfer to the heme dramatically accelerates the Ru-luminescence decay. The crystal structure of a P450(cam):Ru-adamantyl complex reveals access to the active center via a channel whose depth (Ru-Fe distance is 21 A) is virtually the same as that extracted from an analysis of the energy-transfer kinetics. Suitably constructed libraries of sensitizer-linked substrates could be employed to probe the steric and electronic properties of buried active sites.

Camphor 5-Monooxygenase↗

PubMed1995

Stereoselective hydroxylation of norcamphor by cytochrome P450cam. Experimental verification of molecular dynamics simulations.

The stereoselectivity of cytochrome P450cam hydroxylation has been investigated with the enantiomerically pure substrate analog norcamphor. (1R)- and (1S)-norcamphor (> 92 enantiomeric excess) were characterized in the hydroxylation reaction with cytochrome P450cam with respect to the product profile, steady state kinetics, coupling efficiency, and free energy of substrate dissociation. The experimental results demonstrate regiospecificity that is enantiomer-specific and confirm our previously reported prediction that (1R)-norcamphor is hydroxylated preferentially at the 5-carbon and (1S)-norcamphor at the 6-carbon (Bass, M. B., and Ornstein, R. L. (1993) J. Comput. Chem. 14, 541-548); these simulation results are now compared with simulations involving a ferryl oxygen intermediate. Hydroxylation of (1R)-norcamphor was found at the 5-, 6-, and 3-carbons in a ratio of 65:30:5 (respectively), whereas (1S)-norcamphor was oxidized to produce a 28:62:10 ratio of the same products. With the exception of the regiospecificity, all of the reaction and physical parameters are similar for each enantiomer of norcamphor. These results show that the position of the carbonyl group on the hydrocarbon skeleton of norcamphor plays a role in determining the average orientation of this substrate in the active site and suggests that hydrogen bonding interactions can aid in directing the regiospecificity and stereospecificity of the hydroxylation reaction catalyzed by cytochrome P450cam.

Binding Sites↗

PubMed1995

X-ray absorption near edge studies of cytochrome P-450-CAM, chloroperoxidase, and myoglobin. Direct evidence for the electron releasing character of a cysteine thiolate proximal ligand.

The low spin ferric and low and high spin ferrous forms of myoglobin, bacterial cytochrome P-450-CAM, and chloroperoxidase have been examined by Fe-K x-ray absorption edge spectroscopy. The positions of the absorption edge and the shapes of preedge and edge regions of imidazole adducts of ferric P-450-CAM and chloroperoxidase are essentially the same when compared with thiolate-ligated ferric myoglobin. As these three protein derivatives all have six-coordinate, low spin, ferric hemes with axial imidazole and thiolate ligands, the superposition of x-ray absorption edge spectral properties demonstrates that the protein environment does not effect the spectra, provided one compares heme iron centers with identical coordination numbers, spin and oxidation states, and ligand sets. In contrast, a 0.96 eV difference is observed in the energy of the absorption edge for imidazole- and thiolate-ligated ferric myoglobin with the latter shifted to lower energy as observed for ferrous myoglobin states. Similarly, in the low spin ferric-imidazole and ferrous-CO states, the energies of the absorption edge for chloroperoxidase and P-450-CAM are shifted in the direction of the ferrous state (to lower energy) when compared with those for analogous myoglobin derivatives. In the deoxyferrous high spin state, comparison of the edge spectra of chloroperoxidase with analogous data for cytochrome P-450-CAM suggests that the electron density at the iron is similar for these two protein states. The shifts observed in the energies of the x-ray absorption edge for the thiolate-ligated states of these proteins relative to derivatives lacking a thiolate ligand provide a direct measure of the electron releasing character of a thiolate axial ligand. These results therefore support the suggested role of the cysteinate proximal ligand of P-450 as a strong internal electron donor to promote O-O bond cleavage in the putative ferric-peroxide intermediate to generate the proposed ferryl-oxo "active oxygen" state of the reaction cycle.

Camphor 5-Monooxygenase↗

PubMed1995

Identification of nitric oxide synthase as a thiolate-ligated heme protein using magnetic circular dichroism spectroscopy. Comparison with cytochrome P-450-CAM and chloroperoxidase.

Nitric oxide (NO) has recently been recognized as an important biomolecule playing diverse physiological roles. It is synthesized in several different tissues from L-Arg and O2, using NADPH as an electron donor, by a family of heme-containing catalytically self-sufficient monooxygenases known as nitric oxide synthases (NOS). Recently, the CO complex of reduced NOS has been shown to exhibit an absorption maximum near 450 nm, a characteristic spectral feature of cytochrome P-450 (P-450). Yet, the amino acid sequences of NOS and P-450 have no homology. To further probe the active site heme coordination structure and the heme environment of NOS, we have employed magnetic circular dichroism (MCD) and CD spectroscopy in the present study. MCD spectra of several derivatives of rat brain neuronal NOS strikingly resemble those of analogous derivatives of bacterial P-450-CAM and fungal chloroperoxidase, two known thiolate-ligated heme proteins. Given the proven fingerprinting capability of MCD spectroscopy, this provides convincing evidence for endogenous thiolate (cysteinate) ligation to the heme iron of NOS. Furthermore, the heme-related Soret CD bands of NOS (positive) and P-450s (negative), as represented by P-450-CAM, are almost mirror images, whereas chloroperoxidase exhibits totally different CD band shapes. This suggests that the active sites of NOS and P-450 may share some common structural features, but significant distinctions exist between their heme environments in certain aspects such as hydrophobicity or size.

Amino Acid Oxidoreductases↗

PubMed1996

Role of Arg112 of cytochrome p450cam in the electron transfer from reduced putidaredoxin. Analyses with site-directed mutants.

The mechanism for the reduction of ferric cytochrome P450cam by reduced putidaredoxin, the physiological electron donor for the cytochrome, has been studied by using site-directed mutants of cytochrome P450cam, in which Arg112, an amino acid residue at the presumed binding site for putidaredoxin, was changed to several other amino acid residues. The affinity of reduced putidaredoxin for ferric cytochrome P450cam to form a diprotein complex was decreased greatly by changing Arg112 to a neutral amino acid such as Cys, Met, or Tyr. The rate of intracomplex electron transfer from putidaredoxin to cytochrome P450cam also diminished upon replacing the basic residue with neutral ones, being 42, 18, 4.0, 1.3, and 0. 16 s-1 for Arg (wild type), Lys, Cys, Met, and Tyr enzymes, respectively. Furthermore, the oxidation-reduction potential of cytochrome P450cam (Fe3+/Fe2+ couple) decreased in a similar way to the decrease in the rate of electron transfer upon amino acid substitution; the values were -138, -162, -182, -200, and -195 mV for Arg (wild type), Lys, Cys, Met, and Tyr enzymes, respectively. These results indicate that the amino acid substitution at position 112 affects the oxidation-reduction potential of the heme iron in cytochrome P450cam, thereby diminishing the rate of electron transfer between the two metal centers. The rate of electron transfer from putidaredoxin to oxyferrous cytochrome P450cam also diminished upon substitution of Arg112 with a neutral amino acid.

Arginine↗

PubMed2000

A positively charged cluster formed in the heme-distal pocket of cytochrome P450nor is essential for interaction with NADH.

Arg and Lys residues are concentrated on the distal side of cytochrome P450nor (P450nor) to form a positively charged cluster facing from the outside to the inside of the distal heme pocket. We constructed mutant proteins in which the Arg and Lys residues were replaced with Glu, Gln, or Ala. The results showed that this cluster plays crucial roles in NADH interaction. We also showed that some anions such as bromide (Br(-)) perturbed the heme environment along with the reduction step in P450nor-catalyzed reactions, which was similar to the effects caused by the mutations. We determined by x-ray crystallography that a Br(-), termed an anion hole, occupies a key region neighboring heme, which is the terminus of the positively charged cluster and the terminus of the hydrogen bond network that acts as a proton delivery system. A comparison of the predicted mechanisms between the perturbations caused by Br(-) and the mutations suggested that Arg(174) and Arg(64) play a crucial role in binding NADH to the protein. These results indicated that the positively charged cluster is the unique structure of P450nor that responds to direct interaction with NADH.

Amino Acid Sequence↗

PubMed2001

The role of water molecules in the association of cytochrome P450cam with putidaredoxin. An osmotic pressure study.

We have investigated the osmotic pressure dependence of the association between ferric cytochrome P450cam and putidaredoxin (Pdx) to gain an insight into the role of water molecules in the P450cam-reduced Pdx complexation amenable to physiological electron transfer. The association constant was evaluated from the electron transfer rates from reduced Pdx to P450cam. The natural logarithm of the association constant K(a) was linearly reduced by the osmotic pressure, and osmotic stress yields uptake of 25 waters upon association. In contrast, uptake of only 13 waters is observed from the osmotic pressure dependence of the association in the nonphysiological redox partners P450cam and oxidized Pdx. Although general protein-protein associations proceed through dehydration around the complex interface, the interfacial waters could mediate hydrogen-bonding interactions. Therefore, about 10 more interfacial waters imply an additional water-mediated hydrogen-bonding network in the P450cam.reduced Pdx complex, which does not exist in the complex with oxidized Pdx. It is also possible that the water-mediated hydrogen-bonding interactions support a high P450cam affinity for reduced (K(a) = 0.83 microm(-1)) relative to oxidized (K(a) = 0.058 microm(-1)) Pdx. This study points to a novel role of solvents in assisting redox state-dependent interaction between P450cam and Pdx.

Bacterial Proteins↗

PubMed2001

Complex formation of cytochrome P450cam with Putidaredoxin. Evidence for protein-specific interactions involving the proximal thiolate ligand.

We have performed resonance Raman studies on ferrous NO- and CO-adducts of cytochrome P450(cam) and investigated the effects of diprotein complex formation with reduced putidaredoxin. We have found that the Fe-NO stretching mode of NO-P450(cam) can be resolved into two peaks at 551 and 561 cm(-1), and the binding of putidaredoxin increases the intensity of the high frequency component. Because the Fe-NO mode has been shown to be more sensitive to the nature of the heme proximal ligand than to the distal pocket environment, such a perturbation upon putidaredoxin binding is suggestive of changes in conformation or electronic structure that affect the proximal iron-cysteine bond. In accordance with this idea, the isotope shifts for the Fe-XO stretching and Fe-X-O bending modes (X = N or C) are insensitive to the presence or absence of putidaredoxin, indicating that the geometry of the Fe-X-O unit is not significantly altered by the complex formation. On the other hand, complex formation does induce a perturbation of the low frequency heme vibrational modes, suggesting that alterations of the heme electronic structure and/or geometry take place when putidaredoxin binds. We also find that cytochrome b(5) minimally affects the heme active site of the enzyme, although both putidaredoxin and cytochrome b(5) bind to the same or similar site on P450(cam). These observations suggest that there is a key specific interaction between P450(cam) and putidaredoxin, and that this interaction increases the population of a protein conformation that exhibits structural and/or electronic distortions of the heme group associated with the proximal side of the heme pocket and the S --> Fe electron donation. These electronic and structural changes are potentially correlated with H-bonding to the proximal cysteine.

Animals↗

PubMed2002

Crystal structure of the F87W/Y96F/V247L mutant of cytochrome P-450cam with 1,3,5-trichlorobenzene bound and further protein engineering for the oxidation of pentachlorobenzene and hexachlorobenzene.

We reported previously that the F87W/Y96F/V247L mutant of cytochrome P-450cam (CYP101) from Pseudomonas putida catalyzed the rapid oxidation of lightly chlorinated benzenes, but pentachlorobenzene oxidation was slow (Jones, J. P., O'Hare, E. J., and Wong, L. L. (2001) Eur. J. Biochem. 268, 1460-1467). In the present work, we determined the crystal structure of this mutant with bound 1,3,5-trichlorobenzene. The substrate was bound to crystallographically independent CYP101 molecules in at least three different orientations, which were distinguished by the angle between the benzene ring and the porphyrin, and one orientation contained an Fe-Cl interaction. In another orientation, the substrate was almost parallel to the heme, with a C-H bond closest to the iron. The enzyme/substrate contacts suggested that the L244A mutation should promote the binding of pentachlorobenzene and hexachlorobenzene by creating space to accommodate the extra chlorines. The F87W/Y96F/L244A/V247L mutant thus designed was found to oxidize pentachlorobenzene at a rate of 82.5 nmol (nmol CYP101)(-1) min(-1), 45 times faster than the F87W/Y96F/V247L parent mutant. The rate of hexachlorobenzene oxidation was increased 200-fold, to 2.0 min(-1). Both substrates are oxidized to pentachlorophenol, which is degraded by micro-organisms. In principle, the F87W/Y96F/L244A/V247L mutant could have applications in the bioremediation of polychlorinated benzenes.

Amino Acid Substitution↗

PubMed2003

Tyrosine radical formation in the reaction of wild type and mutant cytochrome P450cam with peroxy acids: a multifrequency EPR study of intermediates on the millisecond time scale.

We report a multifrequency (9.6-, 94-, 190-, and 285-GHz) EPR study of a freeze-quenched intermediate obtained from reaction of substrate-free cytochrome P450cam (CYP101) and its Y96F and Y96F/Y75F mutants with peroxy acids. It is generally assumed that in such a shunt reaction an intermediate [Fe(IV)=O, porphyrin-pi-cation radical] is formed, which should be identical to the species in the natural reaction cycle. However, for the wild type as well as for the mutant proteins, a porphyrin-pi-cation radical is not detectable within 8 ms. Instead, EPR signals corresponding to tyrosine radicals are obtained for the wild type and the Y96F mutant. Replacement of both Tyr-96 and Tyr-75 by phenylalanine leads to the disappearance of the tyrosine EPR signals. EPR studies at 285 GHz on freeze-quenched wild type and Y96F samples reveal g tensor components for the radical (stretched g(x) values from 2.0078 to 2.0064, g(y) = 2.0043, and g(z) = 2.0022), which are fingerprints for tyrosine radicals in a heterogeneous polar environment. The measurements at 94 GHz using a fundamental mode microwave resonator setup confirm the 285-GHz study. From the simulation of the hyperfine structure in the 94-GHz EPR spectra the signals have been assigned to Tyr-96 in the wild type and to Tyr-75 in the Y96F mutant. We suggest that a transiently formed Fe(IV)=O porphyrin-pi-cation radical intermediate in P450cam is reduced by intramolecular electron transfer from these tyrosines within 8 ms.

Base Sequence↗

PubMed2004

Refolding processes of cytochrome P450cam from ferric and ferrous acid forms to the native conformation. Formations of folding intermediates with non-native heme coordination state.

Changes in heme coordination state and protein conformation of cytochrome P450(cam) (P450(cam)), a b-type heme protein, were investigated by employing pH jump experiments coupled with time-resolved optical absorption, fluorescence, circular dichroism, and resonance Raman techniques. We found a partially unfolded form (acid form) of ferric P450(cam) at pH 2.5, in which a Cys(-)-heme coordination bond in the native conformation was ruptured. When the pH was raised to pH 7.5, the acid form refolded to the native conformation through a distinctive intermediate. Formations of similar acid and intermediate forms were also observed for ferrous P450(cam). Both the ferric and ferrous forms of the intermediate were found to have an unidentified axial ligand of the heme at the 6th coordination sphere, which is vacant in the high spin ferric and ferrous forms at the native conformation. For the ferrous form, it was also indicated that the 5th axial ligand is different from the native cysteinate. The folding intermediates identified in this study demonstrate occurrences of non-native coordination state of heme during the refolding processes of the large b-type heme protein, being akin to the well known folding intermediates of cytochromes c, in which c-type heme is covalently attached to a smaller protein.

Camphor 5-Monooxygenase↗

PubMed2004

Radical rebound mechanism in cytochrome P-450-catalyzed hydroxylation of the multifaceted radical clocks alpha- and beta-thujone.

Alpha-thujone (1alpha) and beta-thujone (1beta) were used to investigate the mechanism of hydrocarbon hydroxylation by cytochromes P-450(cam) (CYP101) and P-450(BM3) (CYP102). The thujones are hydroxylated by these enzymes at various positions, but oxidation at C-4 gives rise to both rearranged and unrearranged hydroxylation products. Rearranged products result from the formation of a radical intermediate that can undergo either inversion of stereochemistry or ring opening of the adjacent cyclopropane ring. Both of these rearrangements, as well as a C-4 desaturation reaction, are observed. The ring opening clock gives oxygen rebound rates that range from 0.2 x 10(10) to 2.8 x 10(10) s(-1) for the different substrate and enzyme combinations. The C-4 inversion reaction provides independent confirmation of a radical intermediate. The phenol product expected if a C-4 cationic rather than radical intermediate is formed is not detected. The results are consistent with a two-state process and provide support for a radical rebound but not a hydroperoxide insertion mechanism for cytochrome P-450 hydroxylation.

Bacterial Proteins↗

PubMed2005

Reaction of ferric cytochrome P450cam with peracids: kinetic characterization of intermediates on the reaction pathway.

Reactions of substrate-free ferric cytochrome P450cam with peracids to generate Fe=O intermediates have previously been investigated with contradictory results. Using stopped-flow spectrophotometry, the reaction with m-chloroperoxybenzoic acid demonstrated an Fe(IV)=O + porphyrin pi-cation radical (Cpd I) (Egawa, T., Shimada, H., and Ishimura, Y. (1994) Biochem. Biophys. Res. Commun. 201, 1464-1469). By contrast, with peracetic acid, Fe(IV)=O plus a tyrosyl radical were observed by freeze-quench Mossbauer and EPR spectroscopy (Schunemann, V., Jung, C., Trautwein, A. X., Mandon, D., and Weiss, R. (2000) FEBS Lett. 479, 149-154). Our detailed kinetic studies have resolved these contradictory results. At pH >7, a significant fraction of Cpd I is formed transiently, whereas at low pH only a species with a Soret band at 406 nm, presumably Fe(IV)=O + tyrosyl radical, is observed. Evidence for formation of an acylperoxo complex en route to Cpd I was obtained. Because of rapid heme destruction, steps subsequent to formation of the highly oxidized forms could not be fully characterized. Heme destruction was avoided by including peroxidase substrates (e.g. guaiacol), which were oxidized to characteristic peroxidase products as the Fe(III)-P450 was regenerated. Addition of ascorbate to either of the high valent species also reforms the Fe(III) state with only a small loss of heme absorbance. These results indicate that typical peroxidase chemistry occurs with P450cam and offer an explanation for the contrasting results reported earlier. The delineation of improved conditions (pH, temperature, choice of peracid) for generating highly oxidized species with P450cam should be valuable for their further characterization.

Camphor 5-Monooxygenase↗

PubMed2005

Crystallographic study on the dioxygen complex of wild-type and mutant cytochrome P450cam. Implications for the dioxygen activation mechanism.

Two key amino acids, Thr252 and Asp251, are known to be important for dioxygen activation by cytochrome P450cam. We have solved crystal structures of a critical intermediate, the ferrous dioxygen complex (Fe(II)-O2), of the wild-type P450cam and its mutants, D251N and T252A. The wild-type dioxygen complex structure is very much the same as reported previously (Schlichting, I., Berendzen, J., Chu, K., Stock, A. M., Maves, S. A., Benson, D. E., Sweet, R. M., Ringe, D., Petsko, G. A., and Sligar, S. G. (2000) Science 287, 1615-1622) with the exception of higher occupancy and a more ordered structure of the iron-linked dioxygen and two "catalytic" water molecules that form part of a proton relay system to the iron-linked dioxygen. Due to of the altered conformation of the I helix groove these two waters are missing in the D251N dioxygen complex which explains its lower catalytic activity and slower proton transfer to the dioxygen ligand. Similarly, the T252A mutation was expected to disrupt the active site solvent structure leading to hydrogen peroxide formation rather than substrate hydroxylation. Unexpectedly, however, the two "catalytic" waters are retained in the T252A mutant. Based on these findings, we propose that the Thr(252) accepts a hydrogen bond from the hydroperoxy (Fe(III)-OOH) intermediate that promotes the second protonation on the distal oxygen atom, leading to O-O bond cleavage and compound I formation.

Amino Acid Substitution↗

PubMed1998

An ab initio approach to the understanding of cytochrome P450-ligand interactions.

1. We describe the application of novel ab initio quantum mechanical methods to the study of ligand interactions with cytochrome P450cam (CYP101). 2. We find that our techniques accurately describe the transition from a low-spin state to a high-spin state of the haem Fe3+ on binding of a substrate. Furthermore, our methods correctly predict that a large fraction of low-spin character is retained on binding of an inhibitor. 3. We demonstrate the use of 'computational experiments' to elucidate key features of the mechanism of interaction. This leads us to identify a new mechanism for the suppression of the low- to high-spin transition on binding of an inhibitor, namely the shortening of the bond between the Fe atom and the coordinated S atom of the cysteine axial ligand.

Camphor 5-Monooxygenase↗

PubMed1999

First principles investigation of singly reduced cytochrome P450.

1. The application of novel ab initio quantum mechanical methods to the states in the catalytic cycle of cytochrome P450 following the first reduction step is described. 2. A good correlation was found between the calculated energy of reduction and the experimentally determined redox potential for a range of substrate- and substrate analogue-bound systems. 3. On reduction of the haem system, the ground state of Fe remains Fe3+. On binding of a CO molecule, Fe adopts a low-spin Fe2+ state, in agreement with experiment. However, on binding of an O2 molecule, calculations indicate that the system adopts a ferric superoxide ground state, in which the Fe is in a low-spin Fe3+ state.

Camphor 5-Monooxygenase↗

PubMed1992

Protective glove material permeation by organic solids.

A method has been developed for the determination of permeation characteristics of glove materials by organic solids. The system employs a stainless steel exposure cell and allows rapid and uniform contact of either solid disks or powders with minimal membrane bowing. A gas chromatograph equipped with a flame ionization detector was used for monitoring the permeation process, which provided detection limits of 0.9-1.2 ng for the organic solids evaluated. By using an automated system for instrument control and data collection, breakthrough times, steady-state times, and steady-state permeation rates have been determined for five common glove materials when exposed to nine organic solids.

Benzoquinones↗