Effects of Reversing the Protein Positive Charge in the Proximity of the Flavin
N(1) Locus of Choline Oxidase<sup>†</sup>
Mahmoud Ghanem
Giovanni Gadda
10.1021/bi052514m.s001
https://acs.figshare.com/articles/journal_contribution/Effects_of_Reversing_the_Protein_Positive_Charge_in_the_Proximity_of_the_Flavin_N_1_Locus_of_Choline_Oxidase_sup_sup_/3233653
A protein positive charge near the flavin N(1) locus is a distinguishing feature of most
flavoprotein oxidases, with mechanistic implications for the modulation of flavin reactivity. A recent
study showed that in the active site of choline oxidase the protein positive charge is provided by His<sub>466</sub>.
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 flavin N(1) locus. CHO-H466D
formed a stable complex with choline but lost the ability to oxidize the substrate. In contrast to the wild-type enzyme, which binds FAD covalently in a 1:1 ratio, CHO-H466D contained ∼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 flavin 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 ∼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(1) locus,
choline oxidase lost the ability to stabilize negative charges in the active site, irrespective of whether they
develop on the flavin or are borne on ligands, resulting in defective flavinylation of the protein, the decreased
electrophilicity of the flavin, and the consequent loss of catalytic activity.
2006-03-14 00:00:00
charge
choline oxidase
flavin
ability
locus
enzyme
H 466A variant
466D
protein
FAD