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PubMed2005

Photoaffinity labeling of P450Cam by an imidazole-tethered benzophenone probe.

[(3)H]4-Benzoyl-N-[2-(imidazole-4-yl)ethyl]benzamide ([(3)H]HBP) was synthesized and used to photoaffinity label P450(Cam). The imidazole moiety of HBP anchors the compound in the P450(Cam) active site by coordination of the heme iron, thereby insuring that covalent modification occurs in the active site. Additionally, the imidazole anchor provides a known binding orientation of HBP to P450(Cam) from which conclusions about enzyme structure can be drawn based upon the locations of photoadducted residues. Two sites of adduction were identified by MS analysis of digested, photoaffinity labeled P450(Cam). Photoaffinity labeling experiments in the presence of the type II competitive inhibitor, 1-phenylimidazole, were used to assess the specificity of the photoadducts characterized. One adduct was located at Met103 on the flexible B'/C loop region of P450(Cam). The other adduct was localized on the C-helix at Met121. The implications of these data are discussed.

Amino Acid Sequence↗
PubMed2005

Circadian activity rhythms in selectively bred ethanol-preferring and nonpreferring rats.

Chronic alcohol intake is associated with dramatic disruptions in sleep and other circadian biological rhythms in both humans and experimental animals. In human alcoholics, these disruptions persist during extended abstinence and appear to promote relapse to drinking. Whereas chronic ethanol intake alters fundamental properties of the circadian pacemaker in unselected rats, nothing is known concerning circadian pacemaker function in selectively bred ethanol-preferring and nonpreferring rats, which are the most widely accepted animal models of genetic predisposition to alcoholism. The present experiments were designed to characterize free-running circadian activity (wheel-running) rhythms under both constant darkness and constant light in selectively bred ethanol-preferring (P, HAD2) and nonpreferring (NP, LAD2) rats. Differences in circadian organization between ethanol-preferring and nonpreferring animals were seen for both pairs of selected lines (P vs. NP; HAD2 vs. LAD2), but these differences were not identical in the two line pairs. For example, although P rats showed shorter free-running periods than NP rats only in constant light, HAD2 rats showed shorter free-running periods than LAD2 rats only in constant darkness. In addition, ethanol-preferring HAD2 rats showed a high rate of rhythm "splitting" that was not seen in any of the other three lines. Taken together, these results suggest that the circadian pacemakers of P and NP rats differ mainly in light sensitivity, whereas those of HAD2 and LAD2 rats differ in their intrinsic period.

Camphor↗
PubMed2006

FTIR studies of the redox partner interaction in cytochrome P450: the Pdx-P450cam couple.

Recently we have developed a new approach to study protein-protein interactions using Fourier transform infrared spectroscopy in combination with titration experiments and principal component analysis (FTIR-TPCA). In the present paper we review the FTIR-TPCA results obtained for the interaction between cytochrome P450 and the redox partner protein in two P450 systems, the Pseudomonas putida P450cam (CYP101) with putidaredoxin (P450cam-Pdx), and the Bacillus megaterium P450BM-3 (CYP102) heme domain with the FMN domain (P450BMP-FMND). Both P450 systems reveal similarities in the structural changes that occur upon redox partner complex formation. These involve an increase in beta-sheets and alpha-helix content, a decrease in the population of random coil/3(10)-helix structure, a redistribution of turn structures within the interacting proteins and changes in the protonation states or hydrogen-bonding of amino acid carboxylic side chains. We discuss in detail the P450cam-Pdx interaction in comparison with literature data and conclusions drawn from experiments obtained by other spectroscopic techniques. The results are also interpreted in the context of a 3D structural model of the Pdx-P450cam complex.

Bacillus megaterium↗
PubMed2006

Structural analysis of cytochromes P450 shows differences in flexibility of heme 2- and 4-vinyls.

Structural analysis of the orientations of heme vinyl side chains was carried out using the published crystallographic data for different cytochromes P450. Torsional angles (tau, C(alpha)C(beta)-C(a)C(b)) show different distributions for the vinyls in positions 2 and 4. Whereas the orientation of 2-vinyls is rather restricted (tau between -120 degrees and -180 degrees ), the 4-vinyls have a much higher mobility over almost the entire conformational space. On the basis of the empirical correlation recently reported for peroxidases (M.P. Marzocchi, G. Smulevich, Relationship between heme vinyl conformation and the protein matrix in peroxidases, J. Raman Spectrosc. 34 (2003), 725-736), an attempt has been made to compare the observed vinyl orientations with the experimental frequencies of the vinyl stretching vibrational modes. The data for P450 proteins do not exactly match the peroxidase-derived function, although a qualitatively similar relationship is likely to exist. Differences between P450 forms suggest a variability in heme-region flexibility and in communication with the rest of enzyme.

Bacterial Proteins↗
PubMed2003

Compressibility and uncoupling of cytochrome P450cam: high pressure FTIR and activity studies.

The effect of the hydrostatic pressure on the CO ligand stretch vibration in cytochrome P450cam-CO bound with various substrates is studied by FTIR. The vibration frequency is linearily shifted to lower values with increasing pressure. The slope of the shift gives the isothermal compressibility of the heme pocket and is found to be related to the high-spin state content in an opposite direction to that previously observed from the pressure-induced shift of the Soret band. This opposite behaviour is explained by the dual effect of heme pocket water molecules both on the CO ligand and on electrostatic potentials produced by the protein at the distal side. The latter effect disturbs ligand-distal side contacts which are needed for a specific proton transfer in oxygen activation when dioxygen is the ligand. Their loss results in uncoupled H(2)O(2) formation.

Camphor 5-Monooxygenase↗
PubMed2003

Bacterial (CYP101) and mitochondrial P450 systems-how comparable are they?

The bacterial CYP101 system and mitochondrial P450 systems show high similarity. Both systems contain the same protein components, a FAD containing reductase, a ferredoxin of the [2Fe2S] type, and a cytochrome P450. At a first glance they seem to be comparable but there are considerable differences among both proteins. Thus, the ferredoxin components of the two systems display significant structural homology but cannot substitute for each other in functional assays. Going into more detail, pronounced differences between the two systems that affect their biological functions are found.

Amino Acid Sequence↗
PubMed2003

Effector proteins from P450(cam) and methane monooxygenase: lessons in tuning nature's powerful reagents.

Effector proteins alter the kinetic or catalytic course of many oxygenase reactions. One of the first oxygenase effectors to be described was putidaredoxin, which serves to gate electron transfer into oxy-P450(cam). In the nonheme, methane monooxygenase (MMO) system, the B-component (MMOB) serves a distinct effector function by gating substrate and oxygen into the active site of the hydroxylase component (MMOH). Here the binding parameters and binding surfaces of the MMOB-MMOH complex are determined by site-specific labeling, fluorescence titrations, chemical cross-linking, and MALDI-TOF peptide identification. Based on these data, a model for the bimolecular complex is described and a hypothesis for the structural basis for the effector function is elaborated. The bearing on the putidaredoxin effector function is discussed.

Binding Sites↗
PubMed2003

My passion for extreme conditions to solve the cytochrome P450 and NO synthase reaction mechanisms.

Cytochrome P450, and especially its reaction mechanism, has always been a major research interest of Professor I.C. Gunsalus. The reaction cycle of this enzyme is complex, containing many elementary steps and intermediates, and constitutes a challenge for the scientific community. During his repeated stays in our laboratories in Paris and in Montpellier, he contributed decisively to our study of the P450 reaction mechanism under extreme conditions, i.e., at subzero temperatures and at high pressure. From this initial impulse, we continued the work with different forms of cytochrome P450 and later on with nitric oxide synthase. This paper gives an overview of the insights into these enzymes gained by the use of extreme conditions. These exciting achievements were initiated by numerous discussions with Professor Gunsalus and also reflect a long-lasting collaboration and friendship.

Camphor 5-Monooxygenase↗
PubMed2004

Structural and functional characterization of "laboratory evolved" cytochrome P450cam mutants showing enhanced naphthalene oxygenation activity.

To elucidate molecular mechanisms for the enhanced oxygenation activity in the three mutants of cytochrome P450cam screened by 'laboratory evolution' [Nature 399 (1999) 670], we purified the mutants and characterized their functional and structural properties. The electronic absorption and resonance Raman spectra revealed that the structures of heme binding site of all purified mutants were quite similar to that of the wild-type enzyme, although the fraction of the inactivated form, called "P420," was increased. In the reaction with H(2)O(2), only trace amounts of the naphthalene hydroxylation product were detected by gas chromatography. We, therefore, conclude that the three mutants do not exhibit significant changes in the structural and functional properties from those of wild-type P450cam except for the stability of the axial ligand in the reduced form. The enhanced fluorescence in the whole-cell assay would reflect enhancement in the oxygenation activity below the detectable limit of the gas chromatography and/or contributions of other reactions catalyzed by the heme iron.

Amino Acid Substitution↗
PubMed2005

An artificial electron donor supported catalytic cycle of Pseudomonas putida cytochrome P450cam.

Putidaredoxin (PdX), the physiological effector of cytochrome P450cam (P450cam), serves to gate electron transfer into oxy-P450cam during the catalytic cycle of the enzyme. Redox-linked structural changes in PdX are necessary for the effective P450cam turnover reaction. PdX is believed to be difficult to be replaced by an artificial electron donor in the reaction pathway of P450cam. We demonstrate that the catalytic cycle of wild-type P450cam can be supported in the presence of an artificial reductant, potassium ferrocyanide. Upon rapid mixing of ferrocyanide ion with P450cam, we observed an intermediate with spectral features characteristic of compound I. The rate constant for the formation of compound I in the presence of ferrocyanide supported reaction cycle was found to be comparable to the ones observed for H2O2 supported compound I formation in wild-type P450cam, but was much lower than those observed for classical peroxidases. The results presented in this paper form the first kinetic analysis of this intermediate for an artificial electron-driven P450cam catalytic pathway in solution.

Biochemistry↗
PubMed2005

Early years of oxygenase research in Bethesda, Osaka, Urbana, and Kanazawa.

In this brief review, I recollect my experiences of how the studies of pyrocatechase and salicylate hydroxylase led to the isolation and revelation of P450cam. Those experiences were instrumental in the separation, purification, and characterization of the two forms of adrenal cortex mitochondrial P450.

Animals↗
PubMed2005

Thirty years of microbial P450 monooxygenase research: peroxo-heme intermediates--the central bus station in heme oxygenase catalysis.

Oxygen has always been recognized as an essential element of many life forms, initially through its role as a terminal electron acceptor for the energy-generating pathways of oxidative phosphorylation. In 1955, Hayaishi et al. [Mechanism of the pyrocatechase reaction, J. Am. Chem. Soc. 77 (1955) 5450-5451] presented the most important discovery that changed this simplistic view of how Nature uses atmospheric dioxygen. His discovery, the naming and mechanistic understanding of the first "oxygenase" enzyme, has provided a wonderful opportunity and scientific impetus for four decades of researchers. This volume provides an opportunity to recognize the breakthroughs of the "Hayaishi School." Notable have been the prolific contributions of Professor Ishimura et al. [Oxygen and life. Oxygenases, Oxidases and Lipid Mediators, International Congress Series, Elsevier, Amsterdam, 2002], a first-generation Hayaishi product, to characterization of the cytochrome P450 monooxygenases.

Bacterial Proteins↗
PubMed2005

Freeze-quenched iron-oxo intermediates in cytochromes P450.

Since the discovery of cytochromes P450 and their assignment to heme proteins a reactive iron-oxo intermediate as the hydroxylating species has been discussed. It is believed that the electronic structure of this intermediate corresponds to an iron(IV)-porphyrin-pi-cation radical system (Compound I). To trap this intermediate the reaction of P450 with oxidants (shunt pathway) has been used. The common approaches are stopped-flow experiments with UV-visible spectroscopic detection or rapid-mixing/freeze-quench studies with EPR and Mössbauer spectroscopic characterization of the trapped intermediate. Surprisingly, the two approaches seem to give conflicting results. While the stopped-flow data indicate the formation of a porphyrin-pi-cation radical, no such species is seen by EPR spectroscopy, although the Mössbauer data indicate iron(IV) for P450cam (CYP101) and P450BMP (CYP102). Instead, radicals on tyrosine and tryptophan residues are observed. These findings are reviewed and discussed with respect to intramolecular electron transfer from aromatic amino acids to a presumably transiently formed porphyrin-pi-cation radical.

Animals↗
PubMed2004

Anti-AIDS agents. Part 58: synthesis and anti-HIV activity of 1-thia-di-O-(-)-camphanoyl-(+)-cis-khellactone (1-thia-DCK) analogues.

Two 1-thia-DCK analogues (9a and 9b) were synthesized and evaluated for inhibition of HIV-1 replication in H9 lymphocytes. Compound 9a showed excellent anti-HIV activity with an EC(50) value of 0.00012 microM and therapeutic index of 1408000. Compound 9b was less active with EC(50) and TI values of 3.11 microM and 62.3, respectively. The bioassay results indicated that thia-DCK analogues merit attention as potential HIV-1 inhibitors.

Anti-HIV Agents↗
PubMed2004

Anti-AIDS agents. Part 62: anti-HIV activity of 2'-substituted 4-methyl-3',4'-di-O-(-)-camphanoyl-(+)-cis-khellactone (4-methyl DCK) analogs.

Four 4-methyl-3',4'-di-O-(-)-camphanoyl-(+)-cis-khellactone (4-methyl DCK) analogs (7a-d) with different alkyl substituents at the 2'-position were synthesized and evaluated for inhibition of HIV-1 replication in H9 lymphocytes. 2'-Methyl-2'-ethyl-4-methyl DCK (7b) was more potent (EC(50)=0.22 microM, TI>175) than the other three compounds (7a, 7c, and 7d), but significantly less potent than 4-methyl DCK (2, EC(50)=0.0059 microM, TI>6600). The bioassay results indicated that the 2'-substituents had a strong effect on the anti-HIV activity, and gem-dimethyl substitution at the 2'-position was greatly preferable to larger alkyl substituents or hydrogen atoms.

Anti-HIV Agents↗