Kinetic Analyses of the Electron Transfer Mechanism of Cytochrome P450 Reductase and the Role of the Flavin Mononucleotide Semiquinone State

2010 BMPS Research Forum Best Poster Award2nd Place in the 2011 Denman Undergraduate Research Forum2009 SURP Award from the Biochemistry Department2010 Mayer's Award for the OSU College of Biological SciencesCytochrome P450 reductase (CPR) is a mammalian enzyme that plays a crucial role in the metabolism of drugs and other xenobiotics. This diflavin reductase contains two essential flavin cofactors— flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN)—each bound to a separate protein domain. CPR transfers two electrons sequentially from NADPH to the heme-iron of the cytochrome to support its monooxygenase reaction. My research is focused on elucidating CPR’s electron transport mechanism and the role of the FMN semiquinone (FMNsq, one-electron reduced species) in the process. Thus far, my efforts have focused on the Glycine-141 residue in the FMN-binding domain in CPR. This residue is thought to be a critical component in preserving a unique and stabilizing hydrogen bond with the N(5)H group of the neutral FMNsq state. Through the process of site-directed mutagenesis, Glycine-141 has been replaced by a threonine (G141T) and an alanine (G141A, which I personally generated). More recently, this residue has been deleted (G141del) from the loop it occupies in an attempt to alter the conformation of this loop and to disrupt this crucial interaction with the cofactor. Each Glycine-141 alteration is theorized to affect the stability of the FMN semiquinone, as evidenced primarily by changes in the electron transfer between the domains of CPR. The primary goal of my research project, therefore, is to characterize how the alterations in the mutant CPR varieties affect the kinetics of electron transfer. Following the generation of each mutant, the protein was expressed in culture, purified (using affinity chromatography) and finally characterized (using SDS-PAGE analysis, as well as high performance liquid chromatography (HPLC)). Additionally, a series of analytical assays were performed to gain insight into the effects of the Glycine-141 alteration. The first analytical experiments that were performed were steady-state turnover (SSTO) assays, which provide an overall rate of electron transfer through CPR (from the NADPH electron donor to a cytochrome c electron acceptor). Essentially these experiments assessed the activity of the proteins and confirmed, to a certain degree, that all CPR varieties were folded correctly and functioning properly. Upon analyzing the procured turnover data, it was determined that there were, at a cursory level, significant kinetic differences between the Glycine-141 altered mutant CPR variants and wild-type CPR (WT CPR). Oxidative titrations of the CPR varieties with ferricyanide were then performed to establish the level of FMN semiquinone formation among the CPR varieties. These experiments permit for the controlled removal of electron equivalents from the CPR system which allows specific observance of pre-identified redox species of CPR. Again there were significant differences in the amount of semiquinone formed in WT CPR, G141A, G141T, and G141del CPR. The focus of my research then became elucidating the specific mechanistic differences causing these changes in CPR characteristics and how the changes in stability in the FMNsq accounted for these kinetic discrepancies. Two major methods were utilized to elucidate the mechanism of electron transfer in CPR: pre-steady state turnover (pre-SSTO) assays and using iterative exponential and global data fitting along with kinetics modeling software to acquaint our kinetic data to specific biochemical steps. Pre-steady state turnover assays are performed using a stopped-flow spectrophotometer, which allows for a reaction to be monitored within the first couple milliseconds of mixing two solutions, and is therefore a valuable tool in characterizing CPR activity which occurs very quickly. The majority of my research effort is focused on establishing the inter-flavin electron transfer characterization of WT CPR, G141A CPR, G141T CPR and G141del CPR during reductive and oxidative half-reactions in these pre-steady-state turnover assays. Reductive half reactions consist on looking at the transfer of electrons from bound NADPH to the FAD domain of CPR and then from the FAD domain to the FMN domain of CPR. This absorbance plot of the reductive half reaction was observed to be triphasic. The first kinetic phase, which is the rate of hydride transfer from NADPH to the FAD, was not appreciably affected by alterations. However, the first observed species is the disemiquinoid form (each flavin contains one electron) indicating that inter-flavin electron transfer is more rapid. The second phase appears also to be associated with inter-flavin transfer and is dependent on NADP+ and the alteration. In the deletion variant, this phase is associated with the more rapid loss of the disemiquinoid species, consistent with its instability. The third, previously unrecognized, phase was not appreciable affected by the Glycine-141 alterations and may be too slow to be catalytically relevant. The data supports our hypothesis that the unique FMN-binding loop in CPR plays a significant role in its electron-transferring activity and serves to help clarify the specific mechanistic steps in the electron transfer process in diflavin reductases. To complement this reductive half reaction data, oxidative half-reactions, which consist of watching electrons move from the FAD domain to the FMN domain of CPR and finally to a final cytochrome c electron acceptor, were be performed. Effectively, analysis of this oxidative half-reaction experiment allow us to dissect the overall electron transfer from CPR to cytochrome c into individual steps, much like we did with the reductive half reaction. The absorbance change plots from these pre-steady state turnover experiments are often difficult to understand. The most reasonable way to acquaint our kinetic data to specific biochemical events will be to model reaction mechanisms using kinetic simulation software, which allows for the fitting of experimentally acquired data to pre-constructed species-based models. This technique will allow us to make models of the reductive and oxidative half-reactions and relate observed absorbance changes to specific rates of electron transfer between redox species of CPR. After a prototypical model of electron transfer has been made for wild-type CPR, then application of the model to the data of the G141A, G141T and G141del CPR oxidative and reductive half-reactions will allow us to identify more precisely how the alteration in the FMN-binding domain is affecting the electron transfer in CPR. The effectiveness of this modeling software has been proven in our preliminary analysis of the reductive half-reaction data of all CPR varieties, which has generated a better understanding of the experimental data. Our original kinetic model (acquired from literature and experimental inference) for the reduction and interflavin electron transfer of CPR with stoichiometric amounts of NADPH consisted of five species and could account for only the initial reactionary phase in the triphasic reactionary curve seen in the pre-SSTO reductive half-reactions. From this initial construct, I worked methodically building in new species to create a model that would not only allow the KinTek software to fit the kinetics data for WT, G141A, G141T and G141del CPR, but also be able to account for varying reactionary conditions (in particular changing levels of NADP+ in solution, a variable which had been shown to affect the rate of reduction and shape for kinetics traces, presumably due to competitive binding with NADPH to CPR). After some time, we had developed a full model for the 2-election reduction of CPR with NADPH which consisted of nine species. To help fill out this mechanism, additional stopped-flow spectrophotometric pre-SSTO reductive assays were performed on WT CPR and G141del CPR with varying levels of NADP+ and the model was modified to fit these data sets. Currently, our model for the electron transfer in CPR can accurately fit data sets from every CPR variety and could be called roughly complete. From the model, we have already acquired some new insights. Primarily, our model supports the idea that the first observed phase in the reductive half-reaction of CPR is the rate hydride transfer from NADPH. Secondly, the observed rate of the secondary phase of the reactionary trace seems not to be associated with a single electron transfer step, but rather is just the apparent rate of a number of steps and the product of several microscopic rate constants. This secondary phase seems to be the related release of NADP+ from CPR species at varying degrees of flavin reduction (disemiquinoid and FMN fully reduced in particular). In essence, the release of NADP+ creates multiple species which have the same absorption character, allowing for the appearance of a bi- or triphasic curve in the reductive process. Additionally, the rate release of NADP+ appears to be somewhat correlated to the Glycine-141 alteration, an unexpected finding that I am currently investigating. Finally, the competitive binding of NADP+ and NADPH seems to be a significant occurrence as demonstrated by the changes in reactionary trace at high NADP+ concentrations. Seeing the efficacy of the kinetic model and understanding the complex experimental data my current and future research efforts will be to continue working on the reductive half-reaction, as well as, to develop a similar model for the oxidative half-reaction. This process will undoubtedly involve a number of additional clarifying and exploratory experiment to confirm the validity of the theoretical mechanism under differing or untested reaction conditions. However, the final product of this data analysis is a thorough model of electron transfer in cytochrome P450 reductase and upon comparison of electron transfer rates between the CPR mutants; we will have a substantial understanding of the role of the Glycine-141 residue and the FMN semiquinone state in the electron transfer process. The significance of this research project revolve around cytochrome P450 reductase’s role in the oxidative metabolism drugs, xenobiotics, and endogenous compounds in the endoplasmic reticulum of cells. The metabolism of all of these molecules is actually performed by the monooxygenase activity of cytochromes P450, but for these proteins to be enzymatically active, they require the addition of two consecutive electrons. This reduction process is performed primarily by CPR and therefore the electron transfer mechanism that we are studying has implications in better understanding of pharmaceutical design and breakdown, cellular catabolism of toxins and vitamins, and physiological oxidative metabolism as a whole. Additionally, this project could have benefits to the specific biochemical field of flavoprotein research. Cytochrome P450 reductase is a prototypical diflavin reductase enzyme, so mechanistic understanding of this protein could help in the understanding of other diflavin reductases including nitric oxide synthase (NOS), a cancer cell protein (NR1), subunits of bacterial sulfite reductases (SiR), and the flavoprotein domain of flavocytochrome P450BM-3 (which is a bacterial enzyme also studied in the Swenson lab). Although all of these enzymes are different in structure, their operating mechanism cannot be too varied because they electron transfer proceeds through the flavin cofactors. That said, understanding the kinetics of one of these diflavin reductases could augment understanding of this entire class of proteins.A one-year embargo was granted for this item