EVIDENCE THAT THE TRANSFER OF HYDRIDE ION EQUIVALENTS BETWEEN NUCLEOTIDES BY PROTON-TRANSLOCATING TRANSHYDROGENASE IS DIRECT

The molecular masses of the purified, recombinant nucleotide-binding d omains (domains I and III) of transhydrogenase from Rhodospirillum rub rum were determined by electrospray mass spectrometry, The values obta ined, 40,273 and 21,469 Da, for domains I and III, respectively, are s imilar to those estimated from the amino acid sequences of the protein s, Evidently, there are no prosthetic groups or metal centers that can serve as reducible intermediates in hydride transfer between nucleoti des bound to these proteins. The transient-state kinetics of hydride t ransfer catalyzed by mixtures of recombinant domains I and III were st udied by stopped-flow spectrophotometry, The data indicate that oxidat ion of NADPH, bound to domain III, and reduction of acetylpyridine ade nine dinucleotide (an NAD(+) analogue), bound to domain I, are simulta neous and very fast. The transient-state reaction proceeds as a biphas ic burst of hydride transfer before establishment of a steady state, w hich is limited by slow release of NADP(+). Hydride transfer between t he 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 translocatio n.