MODULATION OF THE OXIDATION-REDUCTION POTENTIAL OF THE FLAVIN IN LIPOAMIDE DEHYDROGENASE FROM ESCHERICHIA-COLI BY ALTERATION OF A NEARBY CHARGED RESIDUE, K53R

The epsilon-amino group of a lysine residue occupies a position within bonding distance of the flavin N5 and the bound NADPH pyridinium C4' in glutathione reductase, and it has been suggested that this positive charge influences the redox potential of the FAD [Pai and Schulz (1983) J. Biol. Chem. 258, 1752]. A conserved lysine residue occupies a similar position in lipoamide dehydrogenase. This residue has been replaced by an arginine in lipoamide dehydrogenase from Escherichia coli to give K53R. The spectral and redox properties of the FAD in K53R as well as the interaction of the flavin with bound NAD(+) are profoundly affected by the change. K53R does not catalyze either the dihydrolipoamide-NAD(+) or the NADH-lipoamide reactions except at very low concentrations of the reducing substrate. The absorbance spectrum of K53R in the visible and near-ultraviolet is little changed from that of wild-type enzyme, but in contrast, the spectrum of K53R is sensitive to pH with an apparent pK(a), = 7.0. Unlike the wild-type enzyme, the binding of beta-NAD(+) to K53R alters the spectrum and indicates an apparent K-d = 7.0 mu M at pH 7.6. The flavin fluorescence is partially quenched, and the visible and near-ultraviolet circular dichroism spectrum is changed by beta-NAD(+). K53R is extensively reduced (mostly EH(4)) by 2 equiv of dihydrolipoamide/FAD while the wild-type enzyme is only partially reduced (mostly EH(2)). The rate of this reduction is lowered by approximately 3-fold relative to the wild-type enzyme. Reduction of K53R by NADH at pH 8.5 results in the rapid appearance of a broad band at 750 nm interpreted as an FADH(2)-NAD(+) charge-transfer complex. The rate of the reduction is about half that observed with the wild-type enzyme. Neutral flavin semiquinone appears during photoreduction of K53R. Addition of beta-NAD(+) to the photoreduced enzyme results in the immediate appearance of the FADH(2)-NAD(+) charge-transfer complex. Sensitivity of K53R to four-electron reduction indicates that the redox potential of the FAD has been raised relative to that in the wild-type enzyme, and this was confirmed by direct measurement of the redox potential.