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PubMed1998

Understanding the role of the essential Asp251 in cytochrome p450cam using site-directed mutagenesis, crystallography, and kinetic solvent isotope effect.

Proton transfer in cytochromes P450 is a critical step in the activation of molecular oxygen. Extensive study of the P450cam active site has identified several residues that play a central role in dioxygen bond scission. A highly conserved carboxylate, aspartate-251 in P450cam in the distal helix I, participates in a series of hydrogen-bond/ion pairs near the molecular surface and has been implicated in the catalytic mechanism. Mutation of Asp251 is known to lower activity by 2 orders of magnitude and change the rate-limiting step in the catalytic cycle, suggesting a role for an acid functionality in generation of iron-oxygen reactive intermediates. The turnover rates of the Asp251Asn mutant in various protium-deuterium mixtures have been determined and show a significantly larger kinetic solvent isotope effect, with an overall magnitude of 10 compared to 1.8 for the wild-type P450cam. In addition, a much larger number of protons are involved in the rate-limiting step for the Asp251Asn mutant than in the wild-type enzyme. These results indicate that Asp251 is an essential part of the normal proton delivery machinery required for O-O bond scission. The crystal structure of the Aps251Asn mutant obtained from data collected at cryogenic temperatures has been refined to 1.9 A. Key hydrogen bonds required to hold Asp251 in position have been broken which allows the mutant Asn251 side chain to swing out and away from the O2 binding site leading to a more open active site. This change could allow easier access by water and thus contribute to the observed kinetic solvent isotope effects.

Asparagine↗
PubMed1999

Low-temperature stabilization and spectroscopic characterization of the dioxygen complex of the ferrous neuronal nitric oxide synthase oxygenase domain.

Nitric oxide (NO), an intercellular messenger and an immuno-cytotoxic agent, is synthesized by the family of nitric oxide synthases (NOS), which are thiolate-ligated, heme-containing monooxygenases that convert L-Arg to L-citrulline and NO in a tetrahydrobiopterin (BH4)-dependent manner, using NADPH as the electron donor. The dioxygen complex of the ferrous enzyme has been proposed to be a key intermediate in the NOS catalytic cycle. In this study, we have generated a stable ferrous-O2 complex of the oxygenase domain of rat neuronal NOS (nNOS) by bubbling O2 through a solution of the dithionite-reduced enzyme at -30 degrees C in a cryogenic solvent containing 50% ethylene glycol. The most stable dioxygen complex is obtained using the oxygenase domain which has been preincubated for an extended length of time at 4 degrees C with BH4/dithiothreitol and NG-methyl-L-arginine, a substrate analogue inhibitor. The O2 complex of the nNOS oxygenase domain thus prepared exhibits UV-visible absorption (maxima at 419 and 553 nm, shoulder at approximately 585 nm) and magnetic circular dichroism spectra that are nearly identical to those of ferrous-O2 cytochrome P450-CAM. Our spectral data are noticeably blue-shifted from those seen at 10 degrees C for a short-lived transient species (lambdamax = 427 nm) for the nNOS oxygenase domain using stopped-flow rapid-scanning spectroscopy [Abu-Soud, H. M., Gachhui, R., Raushel, F. M., and Stuehr, D. J. (1997) J. Biol. Chem. 272, 17349], but somewhat similar to those of a relatively stable O2 adduct of L-Arg-free full-length nNOS (lambdamax = 415-416.5 nm) generated at -30 degrees C [Bec, N., Gorren, A. C. F., Voelder, C., Mayer, B., and Lange, R. (1998) J. Biol. Chem. 273, 13502]. Compared with ferrous-O2 P450-CAM, however, the ferrous-O2 adduct of the nNOS oxygenase domain is considerably more autoxidizable and the O2-CO exchange reaction is noticeably slower. The generation of a stable ferrous-O2 adduct of the nNOS oxygenase domain, as described herein, will facilitate further mechanistic and spectroscopic investigations of this important intermediate.

Camphor 5-Monooxygenase↗
PubMed1999

Resonance Raman studies of cytochrome P450BM3 and its complexes with exogenous ligands.

Resonance Raman spectra are reported for both the heme domain and holoenzyme of cytochrome P450BM3 in the resting state and for the ferric NO, ferrous CO, and ferrous NO adducts in the absence and presence of the substrate, palmitate. Comparison of the spectrum of the palmitate-bound form of the heme domain with that of the holoenzyme indicates that the presence of the flavin reductase domain alters the structure of the heme domain in such a way that water accessibility to the distal pocket is greater for the holoenzyme, a result that is consistent with analogous studies of cytochrome P450cam. The data for the exogenous ligand adducts are compared to those previously reported for corresponding derivatives of cytochrome P450cam and document significant and important differences for the two proteins. Specifically, while the binding of substrate induces relatively dramatic changes in the nu(Fe-XY) modes of the ferrous CO, ferric NO, and ferrous NO derivatives of cytochrome P450cam, no significant changes are observed for the corresponding derivatives of cytochrome P450BM3 upon binding of palmitate. In fact, the spectral data for substrate-free cytochrome P450BM3 provide evidence for distortion of the Fe-XY fragment, even in the absence of substrate. This apparent distortion, which is nonexistent in the case of substrate-free cytochrome P450cam, is most reasonably attributed to interaction of the Fe-XY fragment with the F87 phenylalanine side chain. This residue is known to lie very close to the heme iron in the substrate-free derivative of cytochrome P450BM3 and has been suggested to prevent hydroxylation of the terminal, omega, position of long-chain fatty acids.

Bacillus megaterium↗
PubMedDate not supplied

Improving the cytochrome P450 enzyme system for electrode-driven biocatalysis of styrene epoxidation.

Cytochrome P450 enzymes catalyze a vast array of oxidative and reductive biotransformations that are potentially useful for industrial and pharmaceutical syntheses. Factors such as cofactor utilization and slow reaction rates for nonnatural substrates limit their large-scale usefulness. This paper reports several improvements that make the cytochrome P450cam enzyme system more practical for the epoxidation of styrene. NADH coupling was increased from 14 to 54 mol %, and product turnover rate was increased from 8 to 70 min(-1) by introducing the Y96F mutation to P450cam. Styrene and styrene oxide mass balance determinations showed different product profiles at low and high styrene conversion levels. For styrene conversion less than about 25 mol %, the stoichiometry between styrene consumption and styrene oxide formation was 1:1. At high styrene conversion, a second doubly oxidized product, alpha-hydroxyacetophenone, was formed. This was also the exclusive product when Y96F P450cam acted on racemic, commercially available styrene oxide. The alpha-hydroxyacetophenone product was suppressed in reactions where styrene was present at saturating concentrations. Finally, styrene epoxidation was carried out in an electroenzymatic reactor. In this scheme, the costly NADH cofactor and one of the three proteins (putidaredoxin reductase) are eliminated from the Y96F P450cam enzyme system.

Camphor 5-Monooxygenase↗
PubMedDate not supplied

Effect of oxygen on the dehalogenation of 1,2-Dibromo-3-chloropropane by cytochrome P450cam (CYP101).

Cytochromes P450 are known to exhibit diverse catalytic functions against a large number of hydrocarbon substrates. The determinants of specific activity(ies) that operate on specific substrates have not been widely explored. Earlier, we showed that dehalogenation of 1,2-dibromo-3-chloropropane (DBCP) by P450cam (CYP101) monooxygenase exhibits oxygen- and substrate-dependent product distributions and reaction rates (1). Bromochloroacetone was the major conversion product when incubation media were saturated with oxygen, whereas allyl chloride was the sole product accounting for virtually all of the DBCP converted in the absence of oxygen. In an effort to develop a quantitative understanding of the effect of oxygen on product distribution and reaction rate, we have identified first generation products and measured reaction rates at four oxygen levels ranging from 0.01% to 100% saturation. In addition to bromochloroacetone and allyl chloride, a number of bromochloropropene isomers were identified in the presence of oxygen and are thought to be formed by an elimination mechanism. These products accounted for greater than 97 mol % of the reacted DBCP, which was run to high conversion (60-100 mol % DBCP converted). These measurements suggest that P450cam acts on the DBCP substrate through hydroxylation to produce 1-bromo-3-chloroacetone, through reduction to produce allyl chloride, and through elimination to produce bromochloropropene, with oxygen concentration determining the extent of each activity. A global data fitting kinetic model that describes the time-varying concentrations of substrate and products was developed to quantify the controlling level of oxygen on these multiple activities. The parameters of the model were compared with independent measurements and data from the literature.

Camphor 5-Monooxygenase↗
PubMed2006

Novel configurational structures of sodium tetrakis(3-heptafluorobutylryl-(+)-camphorato) Ln(III) complexes with a trapped Na+ by Na+...FC interactions in the solid state and in solution.

For newly prepared sodium tetrakis(3-heptafluorobutylryl-(+)-camphorato) Ln(III) complexes, the X-ray crystal analysis revealed an unprecedented pseudo-achiral dodecahedron (DD-8 (D(2d))), whereas in CHCl3 solution, the exciton CD and 19F NMR spectra showed a novel chiral Delta-SAPR-8 (C4) configuration which is stereospecifically formed by a trapped Na+ ion with Na+...FC (fluorocarbon) interactions.

Camphor↗
PubMed2001

Monooxygenation mechanism by cytochrome p-450.

The substrate oxygenation mechanism by an ultimate species in monooxygenation by cytochrome P-450 (compound I) was investigated by the density functional theory method. An initial model compound was constructed from a structure obtained by 300-ps molecular dynamics simulation of compound I-formed P-450cam under physiologic conditions, and it consisted of porphine for protoporphyrin IX, S(-)-CH(3) for the side chain of Cys357 of the fifth ligand of heme, a methane molecule for the substrate, a heme iron, and an oxygen atom of the sixth ligand of heme. The results of the calculation revealed that the substrate oxygenation mechanism had four elementary processes, i.e., (1) formation of [FeOH](3+) and a substrate radical by hydrogen atom abstraction from the substrate caused by [FeO](3+), (2) rotation of the OH group of the sixth ligand of [FeOH](3+) produced by process 1, (3) substrate radical binding with the [FeOH](3+), and (4) elimination of the oxygenated substrate formed at the sixth ligand binding site. The rate-determining step is process 1, hydrogen atom abstraction from the substrate, and the activation energy was determined to be about 15 kcal/mol. For this reason, it is thought that this reaction occurs in vivo.

Binding Sites↗
PubMed2003

Protein side-chain motion and hydration in proton-transfer pathways. Results for cytochrome p450cam.

Proton-transfer reactions form an integral part of bioenergetics and enzymatic catalysis. The identification of proton-conducting pathways inside a protein is a key to understanding the mechanisms of biomolecular proton transfer. Proton pathways are modeled here as hydrogen bonded networks of proton-conducting groups, including proton-exchanging groups of amino acid side chains and bound water molecules. We focus on the identification of potential proton-conducting pathways inside a protein of known structure. However, consideration of the static structure alone is often not sufficient to detect suitable proton-transfer paths, leading, for example, from the protein surface to the active site buried inside the protein. We include dynamic fluctuations of amino acid side chains and water molecules into our analysis. To illustrate the method, proton transfer into the active site of cytochrome P450cam is studied. The cooperative rotation of amino acids and motion of water molecules are found to connect the protein surface to the molecular oxygen. Our observations emphasize the intrinsic dynamical nature of proton pathways where critical connections in the network may be transiently provided by mobile groups.

Camphor 5-Monooxygenase↗
PubMed2002

Roles of the proximal hydrogen bonding network in cytochrome P450cam-catalyzed oxygenation.

Structural and functional roles of the hydrogen bonding network that surrounds the heme-thiolate coordination of P450(cam) from Pseudomonas putida were investigated. A hydrogen bond between the side chain amide of Gln360 and the carbonyl oxygen of the axial Cys357 was removed in Q360L. The side chain hydrogen bond and the electrostatic interaction between the polypeptide amide proton of Gln360 and the sulfur atom of Cys357 were simultaneously removed in Q360P. The increased electron donation of the axial thiolate in Q360L and Q360P was evidenced by negative shifts of their reduction potentials by 45 and 70 mV, respectively. Together with the results on L358P in which the amide proton at position 358 was removed (Yoshioka, S., Takahashi, S., Ishimori, K., Morishima, I. J. Inorg. Biochem. 2000, 81, 141-151), we propose that the side chain hydrogen bond and the electrostatic interaction of the amide proton with the thiolate ligand cause approximately 45 and approximately 35 mV of positive shifts, respectively, of the redox potential of the heme in P450(cam). The resonance Raman spectra of the ferrous-CO form of the Q360 mutants showed a downshifted Fe-CO stretching mode at 482 approximately 483 cm(-)(1) compared with that of wild-type P450(cam) at 484 cm(-)(1). The Q360 mutants also showed the upshift by 4 approximately 5 cm(-)(1) of the Fe-NO stretching mode in the ferrous-NO form. These Raman results indicate the increase in the sigma-electron donation of the thiolate ligand in the reduced state of the Q360 mutants and were in contrast to the increased pi-back-donation of the thiolate in L358P having an upshifted Fe-CO stretching mode at 489 cm(-)(1). The catalytic activities of the Q360 mutants for the unnatural substrates were similar to those of the wild-type enzyme, indicating that the increased sigma-electron donation does not promote the O-O bond heterolysis in the Q360 mutants, although the increased pi-electron donation in L358P promoted the heterolysis of the O-O bond. We conclude that the functions of the proximal hydrogen bonding network in P450(cam) are to stabilize the heme-thiolate coordination, and to regulate the redox potential of the heme iron. Furthermore, we propose that the pi-electron donation, not the sigma-electron donation, of the thiolate ligand promotes the heterolysis of the O-O bond of dioxygen.

Camphor 5-Monooxygenase↗
PubMed2003

Toxicity screening by electrochemical detection of DNA damage by metabolites generated in situ in ultrathin DNA-enzyme films.

Rapid detection of DNA damage could serve as a basis for in vitro genotoxicity screening for new organic compounds. Ultrathin films (20-40 nm) containing myoglobin or cytochrome P450(cam) and DNA grown layer-by-layer on electrodes were activated by hydrogen peroxide, and the enzyme in the film generated metabolite styrene oxide from styrene. This styrene oxide reacted with double stranded (ds)-DNA in the same film, mimicking metabolism and DNA damage in human liver. DNA damage was detected by square wave voltammetry (SWV) by using catalytic oxidation with Ru(bpy)(3)(2+) (bpy = 2,2'-bipyridine) and by monitoring the binding of Co(bpy)(3)(3+). Damaged DNA reacts more rapidly than intact ds-DNA with Ru(bpy)(3)(3+), giving SWV peaks at approximately 1 V versus SCE that grow larger with reaction time. Co(bpy)(3)(3+) binds more strongly to intact ds-DNA, and its SWV peaks at 0.04 V decreased as DNA was damaged. Little change in SWV signals was found for incubations of DNA/enzyme films with unreactive organic controls or hydrogen peroxide. Capillary electrophoresis and HPLC-MS suggested the formation of styrene oxide adducts of DNA bases under similar reaction conditions in thin films and in solution. The catalytic SWV method was more sensitive than the Co(bpy)(3)(3+) binding assay, providing multiple measurements over a 5 min reaction time.

Biosensing Techniques↗
PubMed2003

Nanosecond photoreduction of cytochrome p450cam by channel-specific Ru-diimine electron tunneling wires.

We report the synthesis and characterization of Ru-diimine complexes designed to bind to cytochrome p450cam (CYP101). The sensitizer core has the structure [Ru(L(2))L'](2+), where L' is a perfluorinated biphenyl bridge (F(8)bp) connecting 4,4'-dimethylbipyridine to an enzyme substrate (adamantane, F(8)bp-Ad), a heme ligand (imidazole, F(8)bp-Im), or F (F(9)bp). The electron-transfer (ET) driving force (-deltaG degrees ) is varied by replacing the ancillary 2,2'-bipyridine ligands with 4,4',5,5'-tetramethylbipyridine (tmRu). The four complexes all bind p450cam tightly: Ru-F(8)bp-Ad (1, K(d) = 0.077 microM); Ru-F(8)bp-Im (2, K(d) = 3.7 microM); tmRu-F(9)bp (3, K(d) = 2.1 microM); and tmRu-F(8)bp-Im (4, K(d) = 0.48 microM). Binding is predominantly driven by hydrophobic interactions between the Ru-diimine wires and the substrate access channel. With Ru-F(8)bp wires, redox reactions can be triggered on the nanosecond time scale. Ru-wire 2, which ligates the heme iron, shows a small amount of transient heme photoreduction (ca. 30%), whereas the transient photoreduction yield for 4 is 76%. Forward ET with 4 occurs in roughly 40 ns (k(f) = 2.8 x 10(7) s(-)(1)), and back ET (Fe(II) --> Ru(III), k(b) approximately 1.7 x 10(8) s(-)(1)) is near the coupling-limited rate (k(max)). Direct photoreduction was not observed for 1 or 3. The large variation in ET rates among the Ru-diimine:p450 conjugates strongly supports a through-bond model of Ru-heme electronic coupling.

Camphor 5-Monooxygenase↗
PubMed2005

Selective solvent interactions in a fluorous reaction system.

Mixtures of chloroform and perfluoro(methylcyclohexane) can be used as solvents for "fluorous" biphase reactions since they exist as two separate phases at low temperature but become a single phase at higher temperatures. Intermolecular nuclear Overhauser effects have been used to investigate the interactions of solvent components with the protons and fluorines of 3-heptafluorobutyrylcamphor in both phases of this biphasic system at 25 degrees C as well as the single phase at 54 degrees C. The results indicate that at 25 degrees C in the perfluorocarbon-rich phase, both solvent components interact with the solute selectively. There are no indications of unusual solute interactions of either solvent component in the chloroform-rich phase and only weak suggestions of selective interactions in the high-temperature phase. Various mechanisms for the enhancement of solute spin-solvent spin cross relaxation rates in the perfluoro(methylcyclohexane)-rich phase are considered. It is suggested that the solvation layer around the solute has a composition and possibly hydrodynamic properties different from those of the bulk solution in this phase. There are indications of appreciable regioselectivity of chloroform interactions with the hydrocarbon part of the solute in all phases.

Camphor↗
PubMed2004

Proton transfer at helium temperatures during dioxygen activation by heme monooxygenases.

In the first measurement of enzymatic proton transfer at liquid helium temperatures, we examine protonation of the peroxo-ferriheme state of heme oxygenase (HO) produced by in situ radiolytic cryoreduction of oxy-HO in H2O and D2O solvents at ca. 4 K and above, and compare these findings with analogous measurements for oxy-P450cam and for oxy-Mb. Proton transfer in HO occurs at helium temperatures in both solvents; it occurs in P450cam at approximately 50 K and higher; in Mb it does not occur until T > 170 K. For Mb, this transfer at 180 K is biphasic, and the majority phase shows a solvent kinetic isotope effect of 3.8. We discuss these results in the context of the picture of environmentally coupled tunneling, which links proton transfer to two classes of protein motions: environmental reorganization (lambda in Marcus-like equations) and protein fluctuations ("active dynamics"; gating) which modulate the distance of proton transfer.

Camphor 5-Monooxygenase↗
PubMed2004

Kinetic isotope effects implicate the iron-oxene as the sole oxidant in P450-catalyzed N-dealkylation.

Multiple oxidants have been implicated as playing a role in cytochrome P450-mediated oxidations. Herein, we report results on N-dealkylation, one of the most facile reactions mediated by P450 enzymes. We have employed the N-oxides of a series of para-substituted 13C2H2-labeled N,N-dimethylanilines to function as both substrates and surrogate oxygen atom donors for P450cam and P4502E1. Kinetic isotope effect profiles obtained using the N-oxide system were found to closely match the profiles produced using the complete NAD(P)H/NAD(P)-P450 reductase/O2 system. The results are consistent with oxidation occurring solely through an iron-oxene species.

Alkylation↗
PubMed2006

QM/MM study of mechanisms for compound I formation in the catalytic cycle of cytochrome P450cam.

In the catalytic cycle of cytochrome P450cam, after molecular oxygen binds as a ligand to the heme iron atom to yield a ferrous dioxygen complex, there are fast proton transfers that lead to the formation of the active species, Compound I (Cpd I), which are not well understood because they occur so rapidly. In the present work, the conversion of the ferric hydroperoxo complex (Cpd 0) to Cpd I has been investigated by combined quantum-mechanical/molecular-mechanical (QM/MM) calculations. The residues Asp(251) and Glu(366) are considered as proton sources. In mechanism I, a proton is transported to the distal oxygen atom of the hydroperoxo group via a hydrogen bonding network to form protonated Cpd 0 (prot-Cpd0: FeOOH(2)), followed by heterolytic O-O bond cleavage that generates Cpd I and water. Although a local minimum is found for prot-Cpd0 in the Glu(366) channel, it is very high in energy (more than 20 kcal/mol above Cpd 0) and the barriers for its decay are only 3-4 kcal/mol (both toward Cpd 0 and Cpd I). In mechanism II, an initial O-O bond cleavage followed by a concomitant proton and electron transfer yields Cpd I and water. The rate-limiting step in mechanism II is O-O cleavage with a barrier of about 13-14 kcal/mol. According to the QM/MM calculations, the favored low-energy pathway to Cpd I is provided by mechanism II in the Asp(251) channel. Cpd 0 and Cpd I are of similar energies, with a slight preference for Cpd I.

Camphor 5-Monooxygenase↗
PubMed2006

Comparing the electronic properties of the low-spin cyano-ferric [Fe(N4)(Cys)] active sites of superoxide reductase and p450cam using ENDOR spectroscopy and DFT calculations.

Superoxide reductase (SOR) and P450 enzymes contain similar [Fe(N)4(SCys)] active sites and, although they catalyze very different reactions, are proposed to involve analogous low-spin (hydro)peroxo-Fe(III) intermediates in their respective mechanisms that can be modeled by cyanide binding. The equatorial FeN4 ligation by four histidine ligands in CN-SOR and the heme in CN-P450cam is directly compared by 14N ENDOR, while the axial Fe-CN and Fe-S bonding is probed by 13C ENDOR of the cyanide ligand and 1Hbeta ENDOR measurements to determine the spin density delocalization onto the cysteine sulfur. There are small, but notable, differences in the bonding between Fe(III) and its ligands in the two enzymes. The ENDOR measurements are complemented by DFT computations that support the semiempirical equation used to compute spin densities on metal-coordinated cysteinyl and shed light on bonding changes as the Fe-C-N linkage bends. They further indicate that H bonds to the cysteinyl thiolate sulfur ligand reduce the spin density on the sulfur in both active sites to a degree that exceeds the difference induced by the alternative sets of "in-plane" nitrogen ligands.

Binding Sites↗