Abstract
The molecular masses of the purified, recombinant nucleotide-binding domains (domains I and III) of transhydrogenase from Rhodospirillum rubrum were determined by electrospray mass spectrometry. The values obtained, 40,273 and 21,469 Da, for domains I and III, respectively, are similar to those estimated from the amino acid sequences of the proteins. Evidently, there are no prosthetic groups or metal centers that can serve as reducible intermediates in hydride transfer between nucleotides bound to these proteins. The transient-state kinetics of hydride transfer catalyzed by mixtures of recombinant domains I and III were studied by stopped-flow spectrophotometry. The data indicate that oxidation of NADPH, bound to domain III, and reduction of acetylpyridine adenine dinucleotide (an NAD+ analogue), bound to domain I, are simultaneous and very fast. The transient-state reaction proceeds as a biphasic burst of hydride transfer before establishment of a steady state, which is limited by slow release of NADP+. Hydride transfer between the nucleotides is evidently direct. This conclusion indicates that the nicotinamide rings of the nucleotides are in close apposition during the hydride transfer reaction, and it imposes firm constraints on the mechanism by which transhydrogenation is linked to proton translocation.
Highlights
Hydride transfer between the nucleotides is direct
The Molecular Weights of Domains I and III of R. rubrum Transhydrogenase Determined by Electrospray Mass Spectrometry—Table I shows the results of an analysis by electrospray mass spectrometry of the molecular masses of the purifed, recombinant domains I and III of R. rubrum transhydrogenase
The similarity between the measured molecular masses of domains I and III, and the values calculated from the amino acid sequences [23], eliminates the possibility of a bound prosthetic group or metal center
Summary
Hydride transfer between the nucleotides is direct. This conclusion indicates that the nicotinamide rings of the nucleotides are in close apposition during the hydride transfer reaction, and it imposes firm constraints on the mechanism by which transhydrogenation is linked to proton translocation. The conclusions from steady-state kinetic analysis of transhydrogenase from various sources (7, 9 –11) have been interpreted as evidence that the reaction proceeds through the formation of a ternary complex of enzyme and nucleotide substrates.
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