Abstract
Transhydrogenase couples the redox reaction between NADH and NADP+ to proton translocation across a membrane. The protein has three components: dI binds NADH, dIII binds NADP+, and dII spans the membrane. Transhydrogenase is a "dimer" of two dI-dII-dIII "monomers"; x-ray structures suggested that the two catalytic sites alternate during turnover. Invariant Tyr146 in recombinant dI of Rhodospirillum rubrum transhydrogenase was substituted with Phe and Ala (proteins designated dI.Y146F and dI.Y146A, respectively). Analytical ultracentrifuge experiments and differential scanning calorimetry show that dI.Y146A more readily dissociates into monomers than wild-type dI. Analytical ultracentrifuge and Trp fluorescence experiments indicate that the dI.Y146A monomers bind NADH much more weakly than dimers. Wild-type dI and dI.Y146F reconstituted activity to dI-depleted membranes with similar characteristics. However, dI.Y146A reconstituted activity in its dimeric form but not in its monomeric form, this despite monomers retaining their native fold and binding to the dI-depleted membranes. It is suggested that transhydrogenase reconstructed with monomers of dI.Y146A is catalytically compromised, at least partly as a consequence of the lowered affinity for NADH, and this results from lost interactions between the nucleotide binding site and the protein beta-hairpin upon dissociation of the dI dimer. The importance of these interactions and their coupling to dI domain rotation in the mechanism of action of transhydrogenase is emphasized. Two peaks in the 1H NMR spectrum of wild-type dI are broadened in dI.Y146A and are tentatively assigned to S-methyl groups of Met resonances in the beta-hairpin, consistent with the segmental mobility of this feature in the structure.
Highlights
We describe the effects of substituting invariant Tyr146, a residue which is centrally located in the dimer interface between the two dI components of R. rubrum transhydrogenase
The dI of R. rubrum transhydrogenase exists as a separate polypeptide
The dI can be removed from inverted membrane vesicles of R. rubrum by centrifugation washing in the absence of NADP(H) [28, 33]; activity is restored to the dII/dIII components in the depleted membranes by adding recombinant dI [26]
Summary
This operation was resuspended in 6 M guanidinium hydrochloride, 20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, Wild-type dI and wild-type dIII from R. rubrum transhy- incubated for 30 min and centrifuged at 100,000 ϫ g for 60 min. To confirm that dI completely refolds under the conditions described, the guanidinium hydrochloride denaturation and subsequent steps were performed with purified wild-type protein. The appearance of the resulting protein on SDS-PAGE was unchanged relative to that from the conventional preparation. It was fully active in reconstitution assays, and the very short wavelength fluorescence emission spectrum was preserved [23]. R. rubrum was grown under photosynthetic conditions and everted membrane vesicles (chromatophores) were prepared and stored as described [28, 29]. Trp fluorescence was measured using a Spex FluoroMax. Isothermal titration calorimetry was carried out using a MicroCal VP-ITC, and data were analyzed using ORIGIN software, as previously described [15]. Acid residues are specified for the R. rubrum transhydrogenase according to the numbering system described [16]
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