Effects of environment on flavin reactivity in morphinone reductase: analysis of enzymes displaying differential charge near the N-1 atom and C-2 carbonyl region of the active-site flavin

The side chain of residue Arg(238) in morphinone reductase (MR) is located close to the N-1/C-2 carbonyl region of the flavin isoalloxazine ring. Duri ng enzyme reduction negative charge develops in this region of the flavin. The positioning of a positively charged side chain in the N-1/C-2 carbonyl region of protein-bound flavin is common to many flavoprotein enzymes. To a ssess the contribution made by Arg(238) in stabilizing the reduced flavin i n MR we isolated three mutant forms of the enzyme in which the position of the positively charged side chain was retracted from the N-1/C-2 carbonyl r egion (Arg(238) --> Lys), the positive charge was removed (Arg(238) --> Met ) or the charge was reversed (Arg(238) --> Glu). Each mutant enzyme retains flavin in its active site. Potentiometric studies of the flavin in the wil dtype and mutant forms of MR indicate that the Ravin semiquinone is not pop ulated to any appreciable extent. Reduction of the flavin in each enzyme is best described by a single Nernst function, and the values of the midpoint reduction potentials (E-12) for each enzyme fall within the region of - 24 7 +/- 10 mV. Stopped-flow studies of NADH binding to wild-type and mutant M R enzymes reveal differences in the kinetics of formation and decay of an e nzyme-NADH charge-transfer complex, reflecting small perturbations in activ e-site geometry. Reduced rates of hydride transfer in the mutant enzymes ar e attributed to altered geometrical alignment of the nicotinamide coenzyme with FMN rather than major perturbations in reduction potential, and this i s supported by an observed entropy-enthalpy compensation effect on the hydr ide transfer reaction throughout the series of enzymes. The data indicate, in contrast with dogma, that the presence of a positively charged side chai n close to the N-1/C-2 carbonyl region of the flavin in MR is not required to stabilize the reduced flavin. This finding may have general implications for flavoenzyme catalysis, since it has generally been assumed that positi ve charge in this region has a stabilizing effect on the reduced form of fl avin.