Directed Mutagenesis of the "GxxS/T" Flavin‐Binding Motif in Cytochrome b5 Reductase

Primary sequence alignments and structural comparisons of members of the flavoprotein transhydrogenase family have identified a four residue sequence corresponding to GRxxST that potentially regulates flavin specificity and incorporation of the prosthetic group. Multiple sequence alignments suggest that specificity is regulated by residue G124 via a G to R substitution. Correct flavin incorporation has also been shown to be modulated by K125 and M126 through hydrogen bonding with the pyrophosphate moiety of FAD. To probe this motif, introduction of a positive charge, charge reversal, and conserved variants were generated for the G124, K125, and M126 residues, respectively, corresponding to G124A/H/K/R, K125A/D/E, and M126C/F/G/P/S/V. Visible CD spectra for each variant displayed an altered conformation of the flavin prosthetic group with the M126P mutant having a reversed polarity in the region of 450 – 500 nm. Dye‐mediated titrations in the presence of NAD+ indicated that that the FAD/FADH2 redox potential was negatively shifted towards that of the potential of free flavin. Differential binding constants obtained revealed that for the G124 variants the FAD cofactor was perturbed so that it allowed for enhanced NAD+ substrate binding in an altered conformation whereas in the M126 mutants binding efficiency was reduced with no spectroscopic changes in the M126P variant.