Cobaltocene-mediated catalytic monooxygenation using holo and heme domain cytochrome P450 BM3

The feasibility of replacing NADPH with 1,1′-dicarboxycobaltocene in the catalytic cycle of cytochrome P450 BM3 has been explored. Using the holoprotein, the surrogate mediator was observed to reduce both the FAD and FMN in the reductase domain, as well as the iron in the heme domain. In an electrochemical system, the mediator was able to support lauric acid hydroxylation at a rate of 16.5 nmol product/nmol enzyme/minute. Similar electron transfer and catalysis were observed for the heme domain alone in the presence of the metallocene; the turnover rate in this case was 1.8 nmol product/nmol enzyme/minute. Parallel studies under the same conditions using a previously reported cobalt sepulchrate mediator showed that the two systems give similar results for both the holoenzyme and the heme domain

Electrochemical and Structural Characterization of Azotobacter vinelandii Flavodoxin II

Azotobacter vinelandii flavodoxin II serves as a physiological reductant of nitrogenase, the enzyme system mediating biological nitrogen fixation. Wildtype A. vinelandii flavodoxin II was electrochemically and crystallographically characterized to better understand the molecular basis for this functional role. The redox properties were monitored on surfactant-modified basal plane graphite electrodes, with two distinct redox couples measured by cyclic voltammetry corresponding to reduction potentials of −483 ± 1 mV and −187 ± 9 mV (vs. NHE) in 50 mM potassium phosphate, 150 mM NaCl, pH 7.5. These redox potentials were assigned as the semiquinone/hydroquinone couple and the quinone/semiquinone couple, respectively. This study constitutes one of the first applications of surfactant-modified basal plane graphite electrodes to characterize the redox properties of a flavodoxin, thus providing a novel electrochemical method to study this class of protein. The X-ray crystal structure of the flavodoxin purified from A. vinelandii was solved at 1.17 Å resolution. With this structure, the native nitrogenase electron transfer proteins have all been structurally characterized. Docking studies indicate that a common binding site surrounding the Fe-protein [4Fe:4S] cluster mediates complex formation with the redox partners Mo-Fe protein, ferredoxin I, and flavodoxin II. This model supports a mechanistic hypothesis that electron transfer reactions between the Fe-protein and its redox partners are mutually exclusive

Protein−Surfactant Film Voltammetry of Wild-Type and Mutant Cytochrome P450 BM3

We are investigating the redox chemistry of wild-type (WT) and mutant (1-12G) cytochrome P450 BM3. Absorption spectra in solution feature the FeIII Soret at 418 nm for WT and a split Soret for 1-12G at 390 and 418 nm. Voltammetry of the proteins within DDAPSS films on the surface of carbon electrodes reveal nearly identical Fe^(III/II) potentials (approximately −200 mV vs Ag/AgCl), but significant differences in k°, 250 vs 30 s^(-1), and Fe^(III/II)−CO potentials, −140 vs −115 mV, for WT vs 1-12G. Catalytic reduction of dioxygen by the proteins on rotating-disk electrodes was analyzed using Levich and Koutecky−Levich treatments. The data reveal 1-12G n and k_(obs) values that are, respectively, 1.7 and 0.07 times those of WT, suggesting that the two proteins differ strikingly in their reactions with dioxygen

Cobaltocene-mediated catalytic monooxygenation using holo and heme domain cytochrome P450 BM3

The feasibility of replacing NADPH with 1,1′-dicarboxycobaltocene in the catalytic cycle of cytochrome P450 BM3 has been explored. Using the holoprotein, the surrogate mediator was observed to reduce both the FAD and FMN in the reductase domain, as well as the iron in the heme domain. In an electrochemical system, the mediator was able to support lauric acid hydroxylation at a rate of 16.5 nmol product/nmol enzyme/minute. Similar electron transfer and catalysis were observed for the heme domain alone in the presence of the metallocene; the turnover rate in this case was 1.8 nmol product/nmol enzyme/minute. Parallel studies under the same conditions using a previously reported cobalt sepulchrate mediator showed that the two systems give similar results for both the holoenzyme and the heme domain

Spectroscopy and Electrochemistry of Cytochrome P450 BM3-Surfactant Film Assemblies

We report analyses of electrochemical and spectroscopic measurements on cytochrome P450 BM3 (BM3) in didodecyldimethylammonium bromide (DDAB) surfactant films. Electronic absorption spectra of BM3−DDAB films on silica slides reveal the characteristic low-spin Fe^(III) heme absorption maximum at 418 nm. A prominent peak in the absorption spectrum of BM3 Fe^(II)−CO in a DDAB dispersion is at 448 nm; in spectra of aged samples, a shoulder at ∼420 nm is present. Infrared absorption spectra of the BM3 Fe^(II)−CO complex in DDAB dispersions feature a time-dependent shift of the carbonyl stretching frequency from 1950 to 2080 cm^(-1). Voltammetry of BM3-DDAB films on graphite electrodes gave the following results: Fe^(III/II) E_(1/2) at −260 mV (vs SCE), ∼300 mV positive of the value measured in solution; ΔS°_(rc), ΔS°, and ΔH° values for water-ligated BM3 in DDAB are −98 J mol^(-1) K^(-1), −163 J mol^(-1) K^(-1), and −47 kJ mol^(-1), respectively; values for the imidazole-ligated enzyme are −8 J mol^(-1) K^(-1), −73 J mol^(-1) K^(-1), and −21 kJ mol^(-1). Taken together, the data suggest that BM3 adopts a compact conformation within DDAB that in turn strengthens hydrogen bonding interactions with the heme axial cysteine, producing a P420-like species with decreased electron density around the metal center

Redox Couples of Inducible Nitric Oxide Synthase

We report direct electrochemistry of the iNOS heme domain in a DDAB film on the surface of a basal plane graphite electrode. Cyclic voltammetry reveals Fe^(III/II) and Fe^(II/I) couples at −191 and −1049 mV (vs Ag/AgCl). Imidazole and carbon monoxide in solution shift the Fe^(III/II) potential by +20 and +62 mV, while the addition of dioxygen results in large catalytic waves at the onset of Fe^(III) reduction. Voltammetry at higher scan rates (with pH variations) reveals that the Fe^(III/II) cathodic peak can be resolved into two components, which are attributable to Fe^(III/II) couples of five- and six-coordinate hemes. Digital simulation of our experimental data implicates water dissociation from the heme as a gating mechanism for ET in iNOS