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Structural basis for differential electron flux in human methionine synthase reductase and cytochrome P450 reductase Meints, Carla Erin


Human diflavin oxidoreductases, methionine synthase reductase (MSR) and cytochrome P450 reductase (CPR), share overall structural organization, substrate specificity, and direction of electron flow. However, MSR exhibits repressed catalytic activity and weak substrate binding compared to CPR. In this study, we identified structural features that control the differential kinetic properties of these flavoenzymes. Single point mutations in the MSR and CPR active sites were studied by steady state and pre-steady state absorbance spectrophotometric techniques to reveal structure-function relationships. In the research first chapter, we investigated the role of a conserved tryptophan in MSR that lies coplanar with the FAD cofactor. Reducing the side chain size resulted in a 1.5- and \textgreater400-fold tighter NADPH binding affinity in W697H and W697S and lowered the preference for NADPH over NADH. W697H also accelerated flavin reduction by 4.6-fold. Thus, the energetic cost of Trp697 displacement by the nicotinamide ring presents an energy barrier to NADPH binding. Consequently, Trp697 regulates coenzyme affinity, and coenzyme preference by ensuring initial recognition of the 2',5'-ADP moiety. The second research chapter evaluated the effect of more conservative mutations, W697Y and W697F, that resulted in improved coenzyme binding affinity and 9-fold accelerated flavin reduction. Equivalent mutations in CPR slowed flavin reduction. Thus, Trp697 gates hydride transfer in MSR, but not in CPR. Instead, following hydride transfer, electron flow in CPR is limited by displacement of the oxidized nicotinamide ring. This is supported by the 46-fold faster flavin reduction of the 2'-phosphate-binding site variant K602AV603K which weakened coenzyme affinity. In the third research chapter, we analyzed the role of FAD proximal His322 which is within hydrogen bonding distance to the catalytic triad residue Asp674 in CPR. The equivalent residue in MSR is Ala312. Through reciprocal mutagenesis, we found that H322A had stronger affinity for NADPH but slower flavin reduction, while A312H weakened coenzyme affinity. His322 is proposed to weaken coenzyme binding affinity by competing with the nicotinamide ring for electrostatic interaction with Asp674, thereby accelerating NADP+ release and electron flow through CPR. The final research chapter shifted to the conserved FAD-stacking tryptophan residue (Trp704) in plant CPR from Artemisia annua (AaCPR) for an additional perspective on overall CPR catalysis. The steady state and pre-steady state data revealed that Trp704 also triggers NADP+ release, however this step is not as critical for electron flux in AaCPR compared to human CPR.

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