The structure and biochemistry of NADH-dependent cytochrome b5 reductase are now consistent

Maria Bewley, C. C. Marohnic, M. J. Barber

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Abstract

Cytochrome b5 reductase (cb5r) (EC 1.6.6.2) catalyzes the reduction of two molecules of cytochrome b5 using NADH as the physiological electron donor. The structure of pig cb5r at 2.4 Å resolution was previously reported in the literature, but it was inconsistent with the biochemistry; for example, K83 and C245 were both implicated in the mechanism, but were not located at the active site. To address this problem, we have determined the structures of cb5r from rat at 2.0 Å resolution and in a complex with NAD+ at 2.3 Å resolution. We found significant differences throughout the rat structure compared to that of pig, including the locations of the lysine and cysteine residues mentioned above. To test the structural models, we made single amino acid substitutions of this lysine and showed that all substitutions produced correctly folded proteins and exhibited normal flavin behavior. However, the apparent kcat(NADH) decreased, and the apparent Km for NADH increased; the Km's for cytochrome b5 were unchanged relative to that of the wild type. The largest effect was for the glutamate-substituted protein, which was further characterized using a charge transfer assay and found to be less efficient at NADH utilization than the wild type. These results are consistent with a role for this lysine in stabilizing the NADH-bound form of cb5r. We have concluded that the pig structure was mistraced in several regions and have reinterpreted mutants in these regions that give rise to the hereditary disease methemoglobinemia.

Original languageEnglish (US)
Pages (from-to)13574-13582
Number of pages9
JournalBiochemistry
Volume40
Issue number45
DOIs
StatePublished - Nov 13 2001

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Cytochrome-B(5) Reductase
Cytochromes b5
Biochemistry
NAD
Cytochrome Reductases
Oxidoreductases
Lysine
Swine
Rats
Substitution reactions
Methemoglobinemia
Inborn Genetic Diseases
Structural Models
Amino Acid Substitution
Cysteine
Charge transfer
Glutamic Acid
Assays
Catalytic Domain
Proteins

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Bewley, Maria ; Marohnic, C. C. ; Barber, M. J. / The structure and biochemistry of NADH-dependent cytochrome b5 reductase are now consistent. In: Biochemistry. 2001 ; Vol. 40, No. 45. pp. 13574-13582.
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The structure and biochemistry of NADH-dependent cytochrome b5 reductase are now consistent. / Bewley, Maria; Marohnic, C. C.; Barber, M. J.

In: Biochemistry, Vol. 40, No. 45, 13.11.2001, p. 13574-13582.

Research output: Contribution to journalArticle

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