Engineering and Characterization of a NADPH-Utilizing Cytochrome b5 Reductase

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Abstract
Microsomal cytochrome b5 reductase (EC 1.6.2.2) catalyzes the reduction of ferricytochrome b5 using NADH as the physiological electron donor. Site-directed mutagenesis has been used to engineer the soluble rat cytochrome b5 reductase diaphorase domain to utilize NADPH as the preferred electron donor. Single and double mutations at residues D239 and F251 were made in a recombinant expression system that corresponded to D239E, S and T, F251R, and Y, D239S/F251R, D239S/F251Y, and D239T/F251R, respectively. Steady-state turnover measurements indicated that D239S/F251Y was bispecific while D239T, D239S/F251R, and D239T/F251R were each NADPH-specific. Wild-type (WT) cytochrome b5 reductase showed a 3700-fold preference for NADH whereas the mutant with the highest NADPH efficiency, D239T, showed an 11-fold preference for NADPH, a 39200-fold increase. Wild-type cytochrome b5 reductase only formed a stable charge-transfer complex with NADH while D239T formed complexes with both NADH and NADPH. The rates of hydride ion transfer, determined by stopped-flow kinetics, were kNADH-WT = 130 s-1, kNADPH-WT = 5 s-1, kNADH-D239T = 180 s-1, and kNADPH-D239T = 73 s-1. Ks determinations by differential spectroscopy demonstrated that D239T could bind nonreducing pyridine nucleotides with a phosphate or a hydroxyl substituent at the 2‘ position, whereas wild-type cytochrome b5 reductase would only bind 2‘ hydroxylated molecules. Oxidation−reduction potentials (E°‘, n = 2) for the flavin cofactor were WT = −268 mV, D239T = −272 mV, WT+NAD+ = −190 mV, D239T+NAD+ = −206 mV, WT+NADP+ = −253 mV, and D239T+NADP+ = −215 mV, which demonstrated the thermodynamic contribution of NADP+ binding to D239T. The crystal structures of D239T and D239T in complex with NAD+ indicated that the loss of the negative electrostatic surface that precluded 2‘ phosphate binding in the wild-type enzyme was primarily responsible for the observed improvement in the use of NADPH by the D239T mutant.