Expression and characterization of a functional canine variant of cytochrome b5 reductase

Abstract

Cytochrome b 5 reductase (c b 5r), a member of the flavoprotein transhydrogenase family of oxidoreductase enzymes, catalyzes the transfer of reducing equivalents from the physiological electron donor, NADH, to two molecules of cytochrome b 5. We have determined the correct nucleotide sequence for the putative full-length, membrane-associated enzyme from Canis familiaris, and have generated a heterologous expression system for production of a histidine-tagged variant of the soluble, catalytic diaphorase domain, comprising residues I33 to F300. Using a simple two-step chromatographic procedure, the recombinant diaphorase domain has been purified to homogeneity and demonstrated to be a simple flavoprotein with a molecular mass of 31,364 ( m/ z) that retained both NADH:ferricyanide reductase and NADH:cytochrome b 5 reductase activities. The recombinant protein contained a full complement of FAD and exhibited absorption and CD spectra comparable to those of a recombinant form of the rat cytochrome b 5 reductase diaphorase domain generated using an identical expression system, suggesting similar protein folding. Oxidation–reduction potentiometric titrations yielded a standard midpoint potential ( E o′) for the FAD/FADH 2 couple of −273 ± 5 mV which was identical to the value obtained for the corresponding rat domain. Thermal denaturation studies revealed that the canine domain exhibited stability comparable to that of the rat protein, confirming similar protein conformations. Initial-rate kinetic studies revealed the canine diaphorase domain retained a marked preference for NADH versus NADPH as reducing substrate and exhibited k cat’s of 767 and 600 s −1 for NADH:ferricyanide reductase and NADH:cytochrome b 5 reductase activities, respectively, with K m’s of 7, 8, and 12 μM for NADH, K 3Fe(CN) 6, and cytochrome b 5, respectively. Spectral-binding constants ( K s) determined for a variety of NAD + analogs indicated the highest and lowest affinities were observed for APAD + ( K s = 71 μM) and PCA + ( K s = >31 mM), respectively, and indicated the binding contributions of the various portions of the pyridine nucleotide. These results provide the first correct sequence for the full-length, membrane-associated form of C. familiaris c b 5r and provide a direct comparison of the enzymes from two phylogenetic sources using identical expression systems that indicate that both enzymes have comparable spectroscopic, kinetic, thermodynamic, and structural properties.