Properties of the soluble polypeptide of the proton-translocating transhydrogenase from Rhodospirillum rubrum obtained by expression in Escherichia coli.
Transhydrogenase, which catalyses the reduction of NADP+ by NADH coupled to proton translocation across a membrane, may be unique in the photosynthetic bacterium Rhodospirillum rubrum. Unlike the homologous enzyme from animal mitochondria and other bacterial sources, it has a water-soluble polypeptide, which exists as a dimer (Ths), that can be reversibly dissociated from the membrane component [Williams, R., Cotton, N. P. J., Thomas, C. M. & Jackson, J. B. (1994) Microbiology, 140, 1595-1604]. We have expressed the gene for Ths in cells of Escherichia coli under control of the tac promoter and a strong ribosome binding site. The protein, purified by column chromatography, fully reconstituted transhydrogenation activity to everted membrane vesicles of Rhs. rubrum that had been washed to remove Ths. The purified expressed protein was prepared in quantities over 100-fold greater than were obtained from wild-type Rhs. rubrum. The fluorescence spectrum of purified expressed Ths had an intense and unusually short wavelength emission maximum at 310 nm with shoulders at 298 and 322 nm. Time-resolved measurements indicated that the fluorescence decay was almost monoexponential with a lifetime of 5.2 ns. On denaturation with 4 M guanidine hydrochloride, the emission band shifted to 352 nm and decreased in intensity. In the native protein, the fluorophore was relatively inaccessible to quenching solutes, such as iodide ions and acrylamide. It is concluded that the fluorescence emission arises mainly from the single tryptophan residue of Ths (Trp72), which is locked into a rigid conformation and is located in highly non-polar environment. The 310-nm fluorescence of Ths was quenched by NADH, maximally to 46%. The apparent binding constant was 18 microM. The fluorescence of Ths-bound NADH was enhanced relative to the nucleotide in free solution and its emission maximum was shifted to a shorter wavelength (440 nm). These data support previous indications that the NADH binding site is located in domain I of proton-translocating transhydrogenase. Excitation of Ths at 280 nm did not lead to sensitized emission at 440 nm from bound NADH. This indicates that the quenching of fluorescence of Ths by NADH does not result from resonance energy transfer from Trp72 to the bound nucleotide. NAD+, NADP+ and NADPH had little effect on the protein fluorescence. The kinetics of quenching of Ths fluorescence by NADH were examined after mixing in a stopped-flow device. The 'on' rate constant for nucleotide binding was approximately 8 x 10(6) M-1 s-1 and the 'off' constant approximately 150 s-1.
Diggle
C
C
School of Biochemistry, University of Birmingham, England.
Hutton
M
M
Jones
G R
GR
Thomas
C M
CM
Jackson
J B
JB
D000595:Amino Acid Sequence; D001692:Biological Transport; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D009250:NADP Transhydrogenases / Q000235:genetics / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism; D055095:Optics and Photonics; D011485:Protein Binding; D011522:Protons; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012247:Rhodospirillum rubrum / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D013050:Spectrometry, Fluorescence
D009711:Nucleotides; D011522:Protons; D011994:Recombinant Proteins; D009250:NADP Transhydrogenases