Heme Domain Of Cytochrome P450 BM3 Research Articles

Electrochemistry of Cytochrome P450 BM3 in Sodium Dodecyl Sulfate Films

Direct electrochemistry of the cytochrome P450 BM3 heme domain (BM3) was achieved by confining the protein within sodium dodecyl sulfate (SDS) films on the surface of basal-plane graphite (BPG) electrodes. Cyclic voltammetry revealed the heme FeIII/II redox couple at -330 mV (vs Ag/AgCl, pH 7.4). Up to 10 V/s, the peak current was linear with the scan rate, allowing us to treat the system as surface-confined within this regime. The standard heterogeneous rate constant determined at 10 V/s was estimated to be 10 s-1. Voltammograms obtained for the BM3-SDS-BPG system in the presence of dioxygen exhibited catalytic waves at the onset of FeIII reduction. The altered heme reduction potential of the BM3-SDS-graphite system indicates that SDS is likely bound in the enzyme active-site region. Compared to other P450-surfactant systems, we find redox potentials and electron-transfer rates that differ by approximately 100 mV and >10-fold, respectively, indicating that the nature of the surfactant environment has a significant effect on the observed heme redox properties.

Reduction of dioxygen catalyzed by pyrene-wired heme domain cytochrome P450 BM3 electrodes.

We have electronically wired the cytochrome P450 BM3 heme domain to a graphite electrode with the use of a pyrene-terminated tether. AFM images clearly reveal that pyrene-wired enzyme molecules are adsorbed to the electrode surface. The enzyme-electrode system undergoes rapid and reversible electron transfer, displaying a standard rate constant higher than that of any other P450-electrode system. We also show that the graphite-pyrene-BM3 system catalyzes the four-electron reduction of dioxygen to water.

Phe393 mutants of cytochrome P450 BM3 with modified heme redox potentials have altered heme vinyl and propionate conformations.

It has been well established that the heme redox potential is affected by many different factors. Among others, it is sensitive to the proximal heme ligand and the conformation of the propionate and vinyl groups. In the cytochrome P450 BM3 heme domain, substitution of the highly conserved phenylalanine 393 results in a dramatic change in the heme redox potential [Ost, T. W. B., Miles, C. S., Munro, A. W., Murdoch, J., Reid, G. A., and Chapman, S. K. (2001) Biochemistry 40, 13421-13429]. We have used resonance Raman spectroscopy to characterize heme structural changes and modification of heme interactions with the protein matrix that are induced by the F393 substitutions and to determine their correlation with the heme redox potential. Our results show that the Fe-S stretching frequency of the 5-coordinated, high-spin ferric heme is not affected by the mutations, suggesting that the electron density in the Fe-S bond in this state is not affected by the F393 mutation and is not a good indicator of the heme redox potential. Substrate binding perturbs the hydrogen bonding between one propionate group and the protein matrix and correlates to both the size of residue 393 and the heme redox potential. However, heme reduction does not affect the conformation of the propionate groups. Although the conformation of the vinyl groups is not affected much by substrate binding, their conformation changes from mainly out-of-plane to predominantly in-plane upon heme reduction. The extent of these conformational changes correlates strongly with the size of the 393 residue and the heme redox potential, suggesting that steric interaction between this residue and the vinyl groups may be of importance in regulating the heme redox potential in the P450 BM3 heme domain. Further implications of our findings for the change in redox potential upon mutation of F393 will be discussed.

Piperidine glycosides targeting the ribosomal decoding site.

Some like it hot: We previously described a laboratory-evolved variant of the cytochrome P450 BM-3 heme domain which functions as a peroxide-driven hydroxylase (peroxygenase, see picture). This biocatalyst does not require additional proteins or NADPH to drive hydroxylation and is amenable to further improvements through protein engineering. Here we describe a thermostable variant whose half-life at 57.5 °C is 50 times that of the F87A variant and 250 times that of the wildtype holoenzyme.

Global incorporation of norleucine in place of methionine in cytochrome P450 BM-3 heme domain increases peroxygenase activity.

In this study we have replaced all 13 methionine residues in the cytochrome P450 BM-3 heme domain (463 amino acids) with the isosteric methionine analog norleucine. This experiment has provided a means of testing the functional limits of globally incorporating into an enzyme an unnatural amino acid in place of its natural analog, and also an efficient way to test whether inactivation during peroxide-driven P450 catalysis involves methionine oxidation. Although there was no increase in the stability of the P450 under standard reaction conditions (in 10 mM hydrogen peroxide), complete substitution with norleucine resulted in nearly two-fold-increased peroxygenase activity. Thermostability was significantly reduced. The fact that the enzyme can tolerate such extensive amino acid replacement suggests that we can engineer enzymes with unique chemical properties via incorporation of unnatural amino acids while retaining or improving catalytic properties. This system also provides a platform for directing enzyme evolution using an extended set of protein building blocks.

A new approach to the study of protein-protein interaction by FTIR: complex formation between cytochrome P450BM-3 heme domain and FMN reductase domain.

We describe a new approach to the study of protein-protein interaction using Fourier transform infrared spectroscopy (FTIR). This approach is based on the combination of FTIR technique with both protein titration experiments and the principal component analysis (factor analysis) of the IR absorption spectra in the 1500-1800 cm(-1) region for the protein mixtures. We have applied this approach to the interaction of the heme domain with the FMN domain of bacterial monooxygenase cytochrome P450BM-3 (CYP102A1). The analysis reveals that the first principal component reflects the protein-protein complex formation because the loading factors show a clear systematic dependence on the concentration of the heme domain according to a titration curve with a dissociation constant of approximately 5 microM. The spectrum of the first principal component has been assigned to structural changes in the secondary structure (increase of beta-sheet and alpha-helix and decrease of turn structures), amino acid side chains (protonation of aspartate and C-terminal COO group), and deprotonation of a propionic acid COOD group in the heme.