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 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.
Venning
J D
JD
School of Biochemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom.
Grimley
R L
RL
Bizouarn
T
T
Cotton
N P
NP
Jackson
J B
JB
D006859:Hydrogen / Q000378:metabolism*; D017136:Ion Transport; D008970:Molecular Weight; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D009250:NADP Transhydrogenases / Q000737:chemistry / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism*; D010084:Oxidation-Reduction; D011522:Protons; D012247:Rhodospirillum rubrum / Q000201:enzymology
D009711:Nucleotides; D011522:Protons; D009249:NADP; D006859:Hydrogen; D009250:NADP Transhydrogenases