Structural basis of inter‐domain electron transfer in human Ncb5or

Abstract

NADH cytochrome b5 oxidoreductase (Ncb5or) is found in all animals and contains three domains: cytochrome b5 (b5), CHORD‐SGT1 (CS) and b5 reductase (b5R). The two redox domains are homologous to microsomal b5 (Cyb5A) and b5R (Cyb5R3), the two known electron donors of stearoyl‐CoA desaturase in vitro. To elucidate the structural basis of inter‐domain electron transfer in human Ncb5or, we generated individual b5 and b5R domains for structural and kinetic studies. The crystal structure of Ncb5or‐b5 was solved by X‐ray diffraction with a resolution of 1.95 Å. There are fewer surface charged residues in Ncb5or‐b5 than Cyb5A, suggesting a weaker electrostatic interaction in b5‐b5R than Cyb5A‐Cyb5R3. This was confirmed in our measurements of electron transfer in b5‐b5R, Cyb5‐Cyb5R3 and mismatched pairs. The linkage through CS likely obviates the need for strong interactions between b5 and b5R in Ncb5or. Ncb5or‐b5 has two helix‐loop‐helix motifs surrounding heme, but differs from Cyb5A in that helix 4 is canted at 45º and shorter. The heme‐ligating His112 residue between helix 3 and 4 is displaced due to one‐residue deletion (before) and insertion (after). Trp114 in helix 4 of Ncb5or‐b5 is a well conserved surface residue potentially involved in inter‐domain recognition or electron transfer. We thus conclude that the Ncb5or‐b5 core has a conserved b5 scaffold with unique features. (Supported by NIH)