Effects of reversing the protein positive charge in the proximity of the flavin N(1) locus of choline oxidase

A protein positive charge near the Ravin N(1) locus is a distinguishing feature of most flavoprotein oxidases, with mechanistic implications for the modulation of Ravin reactivity. A recent study showed that in the active site of choline oxidase the protein positive charge is provided by HiS(466). Here, we have reversed the charge by substitution with aspartate (CHO-H466D) and, for the first time, characterized a flavoprotein oxidase with a negative charge near the Ravin N(1) locus. CHO-H466D formed a stable complex with choline but lost the ability to oxidize the substrate. In contrast to the wildtype enzyme, which binds FAD covalently in a 1:1 ratio, CHO-H466D contained similar to 0.3 FAD per protein, of which 75% was not covalently bound to the enzyme. Anaerobic reduction of CHO-H466D resulted in the formation of a neutral hydroquinone, with no stabilization of the Ravin semiquinone; in contrast, the anionic semiquinone and hydroquinone species were observed with the wild type and a H466A variant of the enzyme. The midpoint reduction potential for the oxidized-reduced couple in CHO-H466D was similar to 160 mV lower than that of the wild-type enzyme. Finally, CHO-H466D lost the ability to form complexes with glycine betaine or sulfite. Thus, with a reversal of the protein charge near the FAD N(l) locus, choline oxidase lost the ability to stabilize negative charges in the active site, irrespective of whether they develop on the Ravin or are borne on ligands, resulting in defective flavinylation of the protein, the decreased electrophilicity of the Ravin, and the consequent loss of catalytic activity.