Premium
Structural basis of Ncb5or, a multi‐domain redox enzyme implicated in diabetes and lipid metabolism
Author(s) -
Deng Bin,
Parthasarathy Sudharsan,
Wang WenFang,
Sturms Ryan,
Hargrove Mark,
Gibney Brian,
Battaile Kevin,
Lovell Scott,
Benson David,
Zhu Hao
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.940.1
Subject(s) - cytochrome b5 , chemistry , electron transfer , redox , heme , electron paramagnetic resonance , enzyme , reductase , biochemistry , photochemistry , nuclear magnetic resonance , inorganic chemistry , physics
NADH cytochrome b5 oxidoreductase (Ncb5or) is a multi‐domain enzyme implicated in diabetes and lipid metabolism. It contains two redox domains that are similar to cytochrome b5 (b5) and its reductase (b5R), a bridging domain with the CS motif, and a novel N‐terminal (N) domain. The heme center has a relatively low redox potential (E 0 = −108 mV). To elucidate the structural basis of inter‐domain electron transfer and properties of the heme redox center, we generated individual domains or various combinations of domains, namely, b5, N‐terminus + b5, b5R and CS+b5R for X‐ray diffraction, kinetic and spectroscopic measurements. A 1.25 Å crystal structure of the b5 domain reveals an orthogonal orientation of the heme‐ligating histidines, consistent with a highly anisotropic low spin EPR signal, the first example of such a heme environment in the b5 superfamily. Kinetic studies of inter‐domain electron transfer suggest an electrostatic “b5‐b5R” interaction which is weaker than in microsomal “Cyb5A‐Cyb5R3” complex, in agreement with fewer charged residues on the surface of b5 than of Cyb5A. This interaction is facilitated by the CS domain and by the N‐terminus, which is shown to be unstructured by CD spectroscopy. Stopped‐flow kinetics suggested a limited role of invariant b5 surface residue Trp 114 in b5R→b5 electron transfer. Thus, Ncb5or employs a unique mechanism for inter‐domain electron transfer. (Supported by NIH)