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1.  Thermal inactivation of the reductase domain of cytochrome P450 BM3 ☆ 
Although the reductase domain of cytochrome P450 BM3 (BMR) catalyzes the reduction of cytochrome c and 2,6-dichlorophe-nolindophenol, we observed a catalytically independent loss of activity. By varying the incubation time for the enzyme prior to reaction initiation, we measured an inactivation rate of 0.22 min−1. We hypothesized that either an active BMR dimer dissociates to an inactive monomer or BMR undergoes denaturation. We were not able to trap or destabilize a dimer, and BMR inactivation proved to be irreversible. Addition of excess FMN only slightly decreased the rate of inactivation from 0.22 to 0.13 min−1, indicating inactivation likely does not reflect loss of flavin. When inactivation rates as a function of temperature were fit to the Arrhenius equation, the energy required to inactivate BMR was 9.9 kcal mol−1—equivalent to a few hydrogen bonds. The potential instability of BMR under certain conditions raises concerns for the use of BMR as a model or surrogate P450 reductase in other systems.
doi:10.1016/j.abb.2005.04.022
PMCID: PMC3664205  PMID: 15950923
Cytochrome; P450; BM3; BMR; Reductase; Thermal stability; Inactivation; Domain
2.  Beta Sheet 2 - Alpha Helix C Loop of Cytochrome P450 Reductase Serves as a Docking Site for Redox Partners 
Biochimica et biophysica acta  2010;1804(6):1285-1293.
As a promiscuous redox partner, the biological role of cytochrome P450 reductase (CPR) depends significantly on protein-protein interactions. We tested a hypothesized CPR docking site by mutating D113, E115, and E116 to alanine and assaying activity toward various electron acceptors as a function of ionic strength. Steady-state cytochrome c studies demonstrated the mutations improved catalytic efficiency and decreased the impact of ionic strength on catalytic parameters when compared to wild type. Based on activity toward 7-ethoxy-4-trifluoro-methylcoumarin, CYP2B1 and CPR favored formation of an active CYP2B1·CPR complex and inactive (CYP2B1)2·CPR complex until higher ionic strength whereby only the binary complex was observed. The mutations increased dissociation constants only for the binary complex and suppressed the ionic strength effect. Studies with a non-binding substrate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) suggest changes in activity toward cytochrome c and CYP2B1 reflect alterations in the route of electron transfer caused by the mutations. Electrostatic modeling of catalytic and binding parameters confirmed the importance of D113 and especially the double mutant E115 and E116 as mediators in forming charge-charge interactions between CPR and complex partners.
doi:10.1016/j.bbapap.2010.02.003
PMCID: PMC2847005  PMID: 20152939
cytochrome P450 reductase; docking; site-directed mutagenesis; CYP2B1; protein-protein interactions; electrostatic interactions
3.  Genome-Wide Analysis of Effectors of Peroxisome Biogenesis 
PLoS ONE  2010;5(8):e11953.
Peroxisomes are intracellular organelles that house a number of diverse metabolic processes, notably those required for β-oxidation of fatty acids. Peroxisomes biogenesis can be induced by the presence of peroxisome proliferators, including fatty acids, which activate complex cellular programs that underlie the induction process. Here, we used multi-parameter quantitative phenotype analyses of an arrayed mutant collection of yeast cells induced to proliferate peroxisomes, to establish a comprehensive inventory of genes required for peroxisome induction and function. The assays employed include growth in the presence of fatty acids, and confocal imaging and flow cytometry through the induction process. In addition to the classical phenotypes associated with loss of peroxisomal functions, these studies identified 169 genes required for robust signaling, transcription, normal peroxisomal development and morphologies, and transmission of peroxisomes to daughter cells. These gene products are localized throughout the cell, and many have indirect connections to peroxisome function. By integration with extant data sets, we present a total of 211 genes linked to peroxisome biogenesis and highlight the complex networks through which information flows during peroxisome biogenesis and function.
doi:10.1371/journal.pone.0011953
PMCID: PMC2915925  PMID: 20694151
4.  Global Analysis of Protein-Protein Interactions Reveals Multiple Cytochrome P450 2E1–Reductase Complexes 
Biochemistry  2007;46(35):10192-10201.
Although a single binary functional complex between cytochrome P450 and cytochrome P450 reductase (CPR) has been generally accepted in the literature, this simple model failed to explain experimentally observed catalytic activity of recombinant P450 2E1 in dependence on the total concentration of added CPR-K56Q mutant. Our rejection of the simplest 1:1 binding model was based on two independent lines of experimental evidence. First, under the assumption of the 1:1 binding model, separate analyses of titration curves obtained while varying either P450 or CPR concentrations individually produced contradictory results. Second, an asymmetric Job plot suggested the existence of higher order molecular complexes. To identify the most probable complexation mechanism, we generated a comprehensive data set where the concentrations of both P450 and P450 were varied simultaneously, rather than one at a time. The resulting two-dimensional data were globally fit to 32 candidate mechanistic models, involving the formation of binary, ternary, and quaternary P450:CPR complexes, in the absence or presence or P450 and CPR homodimers. Of the 32 candidate models (mechanisms), two models were approximately equally successful in explaining our experimental data. The first plausible model involves the binary complex P450•CPR, the quaternary complex (P450)2• (CPR)2, and the homodimer (P450)2. The second plausible model additionally involves a weakly bound ternary complex (P450)2•CPR. Importantly, only the binary complex P450•CPR seems catalytically active in either of the two most probable mechanisms.
doi:10.1021/bi7003476
PMCID: PMC2592557  PMID: 17685587
5.  CYP2E1 Active Site Residues in Substrate Recognition Sequence 5 Identified by Photoaffinity Labeling and Homology Modeling 
Despite its biological importance, our knowledge of active site structure and relevance of critical amino acids in CYP2E1 catalytic processes remain limited. In this study, we identified CYP2E1 active site residues using photoaffinity labeling with 7-azido-4-methylcoumarin (AzMC) coupled with a CYP2E1 homology model. In the absence of light, AzMC was an effective competitor against substrate p-nitrophenol oxidation by CYP2E1. Photoactivation of AzMC led to a concentration-dependent loss in CYP2E1 activity and structural integrity resulting from the modification of both heme and protein. The photolabeling reaction degraded heme and produced a possible heme adduct. Probe incorporation into the protein occurred at multiple sites within substrate recognition sequence 5 (SRS-5). Based on a CYP2E1 homology model, we hypothesize AzMC labels SRS-5 residues, Leu363, Val364 and Leu368, in the active site. In addition, we propose a series of phenylalanines, especially Phe106, mediate contacts with the coumarin.
doi:10.1016/j.abb.2006.10.028
PMCID: PMC1994253  PMID: 17222385
cytochrome; P450 2E1; CYP2E1; photoaffinity; labeling; modeling; active site; azido

Results 1-5 (5)