Sulfite Binding to a Flavodehydrogenase, Cytochrome b2 from Baker's Yeast

Abstract

Baker''s yeast L-lactate dehydrogenase [EC 1.1.2.3] (flavocytochrome .beta.2) is a typical flavodehydrogenase, in that it accepts 2 electrons from the substrate but has a monoelectronic acceptor. Yet it forms a red semiquinone and it is shown in this paper that it forms a reversible covalent complex with sulfite (Kd = 1.4 .mu.M). This complex can be observed by difference spectroscopy and provides a convenient tool for visualizing the flavin chromophore, usually hidden behind the intense heme absorbance. A number of anions (D-lactate, oxalate and pyruvate) are inhibitors of the enzymatic reaction and induce spectral perturbations of the flavin spectrum. Probably 2 positive charges exist at the active site: one which stabilizes the red semiquinone and one which attracts organic anions and sulfite. Apparently the correlation between reactivity with sulfite and reactivity with O2 among flavoproteins may not be as general as previously proposed.