Study of the individual cytochrome b5 and cytochrome b5 reductase domains of Ncb5or reveals a unique heme pocket and a possible role of the CS domain.
NADH cytochrome b(5) oxidoreductase (Ncb5or) is found in animals and contains three domains similar to cytochrome b(5) (b(5)), CHORD-SGT1 (CS), and cytochrome b(5) reductase (b(5)R). Ncb5or has an important function, as suggested by the diabetes and lipoatrophy phenotypes in Ncb5or null mice. To elucidate the structural and functional properties of human Ncb5or, we generated its individual b(5) and b(5)R domains (Ncb5or-b(5) and Ncb5or-b(5)R, respectively) and compared them with human microsomal b(5) (Cyb5A) and b(5)R (Cyb5R3). A 1.25 Å x-ray crystal structure of Ncb5or-b(5) reveals nearly orthogonal planes of the imidazolyl rings of heme-ligating residues His(89) and His(112), consistent with a highly anisotropic low spin EPR spectrum. Ncb5or is the first member of the cytochrome b(5) family shown to have such a heme environment. Like other b(5) family members, Ncb5or-b(5) has two helix-loop-helix motifs surrounding heme. However, Ncb5or-b(5) differs from Cyb5A with respect to location of the second heme ligand (His(112)) and of polypeptide conformation in its vicinity. Electron transfer from Ncb5or-b(5)R to Ncb5or-b(5) is much less efficient than from Cyb5R3 to Cyb5A, possibly as a consequence of weaker electrostatic interactions. The CS linkage probably obviates the need for strong interactions between b(5) and b(5)R domains in Ncb5or. Studies with a construct combining the Ncb5or CS and b(5)R domains suggest that the CS domain facilitates docking of the b(5) and b(5)R domains. Trp(114) is an invariant surface residue in all known Ncb5or orthologs but appears not to contribute to electron transfer from the b(5)R domain to the b(5) domain.
Deng
Bin
B
Department of Clinical Laboratory Sciences, University of Kansas Medical Center, Kansas City, Kansas 66160, USA.
Parthasarathy
Sudharsan
S
Wang
WenFang
W
Gibney
Brian R
BR
Battaile
Kevin P
KP
Lovell
Scott
S
Benson
David R
DR
Zhu
Hao
H
D000818:Animals; D018360:Crystallography, X-Ray; D042966:Cytochrome-B(5) Reductase / Q000737:chemistry* / Q000235:genetics; D015786:Cytochromes b5 / Q000737:chemistry* / Q000235:genetics; D006418:Heme / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D040681:Structural Homology, Protein
D006418:Heme; D015786:Cytochromes b5; C488723:Ncb5or protein, mouse; D042966:Cytochrome-B(5) Reductase