The oxidation of alcohols to aldehydes is catalyzed by a number of flavin-dependent enzymes, which have been grouped in the glucose-methanol-choline oxidoreductase enzyme superfamily. These enzymes exhibit little sequence similarity in their substrates binding domains, but share a highly conserved catalytic site, suggesting a similar activation mechanism for the oxidation of their substrates. In this study, the fully conserved histidine residue at position 466 of choline oxidase was replaced with an alanine residue by site-directed mutagenesis and the biochemical, spectroscopic, and mechanistic properties of the resulting CHO-H466A mutant enzyme were characterized. CHO-H466A showed k(cat) and k(cat)/K(m) values with choline as substrate that were 60- and 1000-fold lower than the values for the wild-type enzyme, while the k(cat)/K(m) value for oxygen was unaffected, suggesting the involvement of His(466) in the oxidation of the alcohol substrate but not in the reduction of oxygen. Replacement of His(466) with alanine significantly affected the microenvironment of the flavin, as indicated by the altered behavior of CHO-H466A with sulfite and dithionite. In agreement with this conclusion, a midpoint reduction potential of +106 mV for the two-electron transfer in the catalytically competent enzyme-product complex was determined at pH 7 for CHO-H466A, which was approximately 25 mV more negative than that of the wild-type enzyme. Enzymatic activity in CHO-H466A could be partially rescued with exogenous imidazolium, but not imidazole, consistent with the protonated form of histidine exerting a catalytic role. pH profiles for glycine betaine inhibition, the deprotonation of the N(3)-flavin locus, and the k(cat)/K(m) value for choline all showed a significant shift upward in their pK(a) values, consistent with a change in the polarity of the active site. Finally, kinetic isotope effects with isotopically labeled substrate and solvent indicated that the histidine to alanine substitution affected the timing of substrate OH and CH bond cleavages, consistent with removal of the hydroxyl proton being concerted with hydride transfer in the mutant enzyme. All taken together, the results presented in this study suggest that in choline oxidase, His(466) modulates the electrophilicity of the enzyme-bound flavin and the polarity of the active site, and contributes to the stabilization of the transition state for the oxidation of choline to betaine aldehyde.

Choline oxidase catalyzes the four-electron oxidation of choline to glycine betaine, with molecular oxygen acting as primary electron acceptor. Recently, the recombinant enzyme expressed in Escherichia coli was purified to homogeneity and shown to contain FAD in a mixture of oxidized and anionic semiquinone redox states [Fan et al. (2003) Arch. Biochem. Biophys., in press]. In this study, methods have been devised to convert the enzyme-bound flavin semiquinone to oxidized FAD and vice versa, allowing characterization of the resulting forms of choline oxidase. The enzyme-bound oxidized flavin showed typical UV-vis absorbance peaks at 359 and 452 nm (with epsilon(452) = 11.4 M(-1) cm(-1)) and emitted light at 530 nm (with lambda(ex) at 452 nm). The affinity of the enzyme for sulfite was high (with a K(d) value of approximately 50 microM at pH 7 and 15 degrees C), suggesting the presence of a positive charge near the N(1)C(2)=O locus of the flavin. The enzyme-bound anionic flavin semiquinone was unusually insensitive to oxygen or ferricyanide at pH 8 and showed absorbance peaks at 372 and 495 nm (with epsilon(372) = 19.95 M(-1) cm(-1)), maximal fluorescence emission at 454 nm (with lambda(ex) at 372 nm), circular dichroic signals at 370 and 406 nm, and an ESR peak-to-peak line width of 13.9 G. Both UV-vis absorbance studies on the enzyme under turnover with choline and steady-state kinetic data with either choline or betaine aldehyde were consistent with the flavin semiquinone being not involved in catalysis. The pH dependence of the kinetic parameters at varying concentrations of both choline and oxygen indicated that a catalytic base is required for choline oxidation but not for oxygen reduction and that the order of the kinetic steps involving substrate binding and product release is not affected by pH.

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It has been found that with mutation of two surface residues (Lys(22) --> Glu and His(104) --> Arg) in human purine nucleoside phosphorylase (hPNP), there is an enhancement of catalytic activity in the chemical step. This is true although the mutations are quite remote from the active site, and there are no significant changes in crystallographic structure between the wild-type and mutant active sites. We propose that dynamic coupling from the remote residues to the catalytic site may play a role in catalysis, and it is this alteration in dynamics that causes an increase in the chemical step rate. Computational results indicate that the mutant exhibits stronger coupling between promotion of vibrations and the reaction coordinate than that found in native hPNP. Power spectra comparing native and mutant proteins show a correlation between the vibrations of Immucillin-G (ImmG):O5'...ImmG:N4' and H257:Ndelta...ImmG:O5' consistent with a coupling of these motions. These modes are linked to the protein promoting vibrations. Stronger coupling of motions to the reaction coordinate increases the probability of reaching the transition state and thus lowers the activation free energy. This motion has been shown to contribute to catalysis. Coincident with the approach to the transition state, the sum of the distances of ImmG:O4'...ImmG:O5'...H257:Ndelta became smaller, stabilizing the oxacarbenium ion formed at the transition state. Combined results from crystallography, mutational analysis, chemical kinetics, and computational analysis are consistent with dynamic compression playing a significant role in forming the transition state. Stronger coupling of these pairs is observed in the catalytically enhanced mutant enzyme. That motion and catalysis are enhanced by mutations remote from the catalytic site implicates dynamic coupling through the protein architecture as a component of catalysis in hPNP.

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(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 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 approximately 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 approximately 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.