Heterologous expression of an endogenous rat cytochrome b(5)/cytochrome b(5) reductase fusion protein: identification of histidines 62 and 85 as the heme axial ligands

Archives of Biochemistry and Biophysics
C Ainsley DavisM J Barber

Abstract

The gene coding for expression of an endogenous soluble fusion protein comprising a b-type cytochrome-containing domain and a FAD-containing domain has been cloned from rat liver mRNA. The 1461-bp hemoflavoprotein gene corresponded to a protein of 493 residues with the heme- and FAD-containing domains comprising the amino and carboxy termini of the protein, respectively. Sequence analysis indicated the heme and flavin domains were directly analogous to the corresponding domains in microsomal cytochrome b(5) (cb5) and cytochrome b(5) reductase (cb5r), respectively. The full-length fusion protein was purified to homogeneity and demonstrated to contain both heme and FAD prosthetic groups by spectroscopic analyses and MALDI-TOF mass spectrometry. The cb5/cb5r fusion protein was able to utilize both NADPH and NADH as reductants and exhibited both NADPH:ferricyanide (k(cat) = 21.7 s(-1), K(NADPH)(m) = 1 microM. K(FeCN6)(m) = 8 microM) and NADPH:cytochrome c (k(cat) = 8.3 s(-1), K(NADPH)(m) = 1 microM. K(cyt c)(m) = 7 microM) reductase activities with a preference for NADPH as the reduced pyridine nucleotide substrate. NADPH-reduction was stereospecific for transfer of the 4R-proton and involved a hydride transfer mechanism with a kin...Continue Reading

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Citations

May 8, 2004·The Journal of Biological Chemistry·Hao ZhuH Franklin Bunn
Jul 13, 2004·Proceedings of the National Academy of Sciences of the United States of America·Jianxin XieH Franklin Bunn
Nov 1, 2008·Molecular and Cellular Neurosciences·Alejandro K Samhan-AriasCarlos Gutierrez-Merino
Nov 2, 2012·Critical Reviews in Biotechnology·Fatemeh ElahianSeyed Abbas Mirzaei
Nov 11, 2008·Biochemistry. Biokhimii︠a︡·A V YantsevichS A Usanov
Jun 11, 2003·The Journal of Biological Chemistry·Frederik A J RotsaertMichael H Gold
Sep 25, 2003·Biochemistry·Christopher C MarohnicMichael J Barber

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