Protein fluorescence and electronic energy transfer in the determination of molecular dimensions and rotational relaxation times of native and coenzyme-bound horse liver alcohol dehydrogenase.

The intrinsic fluorescence lifetimes of horse liver alcohol dehydrogenase (EC 1.1.1.1) and pig heart isocitrate dehydrogenase (EC 1.1.1.42) have been determined to be 5.36 ns and 4.84 ns, respectively. When reduced coenzyme is bound, the fluorescence lifetime of alcohol dehydrogenase is reduced to 4.98 ns while that of isocitrate dehydrogenase remains unchanged. Oxidized coenzymes have no effect on fluorescence lifetimes of alcohol and isocitrate dehydrogenases. This virtual constancy of protein fluorescence lifetimes has allowed the conclusion to be reached that in protein-ligand complexes with equilibrium constants in the range of 10(4)-10(6) M(-1), the static mode of quenching is substantial. The observation of resonance energy transfer in alcohol dehydrogenase-NADH complex facilitates the determination of the distance between tryptophan and the reduced nicotinamide ring involved in the transfer as 30.6 A, compared to the effective molecular radius of 36.2 A for alcohol dehydrogenase. The increased rotational relaxation times of coenzyme-bound alcohol dehydrogenase relative to the unliganded form (sigmah = 72 ns) indicate in this protein structural fluctuations occurring in the time range of nanoseconds.