Arginine 91 is not essential for flavin incorporation in hepatic cytochrome b(5) reductase

Archives of Biochemistry and Biophysics
Christopher C Marohnic, Michael J Barber

Abstract

Cytochrome b(5) reductase (cb5r) catalyzes the transfer of reducing equivalents from NADH to cytochrome b(5). Utilizing an efficient heterologous expression system that produces a histidine-tagged form of the hydrophilic, diaphorase domain of the enzyme, site-directed mutagenesis has been used to generate cb5r mutants with substitutions at position 91 in the primary sequence. Arginine 91 is an important residue in binding the FAD prosthetic group and part of a conserved "RxY(T)(S)xx(S)(N)" sequence motif that is omnipresent in the "ferredoxin:NADP(+) reductase" family of flavoproteins. Arginine 91 was replaced with K, L, A, P, D, Q, and H residues, respectively, and all the mutant proteins purified to homogeneity. Individual mutants were expressed with variable efficiency and all exhibited molecular masses of approximately 32 kDa. With the exception of R91H, all the mutants retained visible absorption spectra typical of a flavoprotein, the former being produced as an apoprotein. Visible absorption spectra of R91A, L, and P were red shifted with maxima at 458 nm, while CD spectra indicated an altered FAD environment for all the mutants except R91K. Fluorescence spectra showed a reduced degree of intrinsic flavin fluorescence que...Continue Reading

References

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Citations

Nov 2, 2012·Critical Reviews in Biotechnology·Fatemeh ElahianSeyed Abbas Mirzaei
Oct 26, 2001·Protein Expression and Purification·M J Barber, G B Quinn
Jul 18, 2018·Applied Microbiology and Biotechnology·Sreeahila Retnadhas, Sathyanarayana N Gummadi
Jun 8, 2002·Archives of Biochemistry and Biophysics·Michael J BarberVeronica V Pollock
Jun 25, 2003·Protein Expression and Purification·C Ainsley Davis, Michael J Barber
Oct 19, 2004·Archives of Biochemistry and Biophysics·C Ainsley DavisMichael J Barber
Jul 4, 2006·Archives of Biochemistry and Biophysics·Glenn W RomaMichael J Barber

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