Abstract
Transhydrogenase undergoes conformational changes to couple the redox reaction between NAD(H) and NADP(H) to proton translocation across a membrane. The protein comprises three components: dI, which binds NAD(H); dIII, which binds NADP(H); and dII, which spans the membrane. Experiments using isothermal titration calorimetry, analytical ultracentrifugation, and small angle x-ray scattering show that, as in the crystalline state, a mixture of recombinant dI and dIII from Rhodospirillum rubrum transhydrogenase readily forms a dI(2)dIII(1) heterotrimer in solution, but we could find no evidence for the formation of a dI(2)dIII(2) tetramer using these techniques. The asymmetry of the complex suggests that there is an alternation of conformations at the nucleotide-binding sites during proton translocation by the complete enzyme. The characteristics of nucleotide interaction with the isolated dI and dIII components and with the dI(2)dIII(1) heterotrimer were investigated. (a) The rate of release of NADP(+) from dIII was decreased 5-fold when the component was incorporated into the heterotrimer. (b) The binding affinity of one of the two nucleotide-binding sites for NADH on the dI dimer was decreased about 17-fold in the dI(2)dIII(1) complex; the other binding site was unaffected. These observations lend strong support to the alternating-site mechanism.
Highlights
Transhydrogenase, found in the cytoplasmic membranes of bacteria, and in the inner membranes of animal mitochondria, couples the redox reaction between NAD(H) and NADP(H) to the translocation of protons
On the basis of the crystal structures and the observations on the behavior of the complex of dI and dIII in solution, we proposed an alternating-site model for intact transhydrogenase; conformational changes resulting from proton translocation across the membrane give rise to changes in interaction between dIII and dI alternately in the two halves of the molecule, and these drive the nucleotide-binding changes that are
The Solution Stability of the dI2dIII1 Heterotrimer of Transhydrogensase—Experiments with the isothermal titration calorimeter showed that the binding of R. rubrum dI to R. rubrum dIII is accompanied by an enthalpy change of ϩ9.48 kJ1⁄7molϪ1 of dIII
Summary
Its function in energy metabolism, biosynthesis, and detoxification has been discussed at length [1, 2]. The dI and dIII components of transhydrogenases can be separately expressed and isolated as stable, water-soluble proteins, which bind their cognate nucleotides. On the basis of the crystal structures and the observations on the behavior of the complex of dI and dIII in solution, we proposed an alternating-site model for intact transhydrogenase; conformational changes resulting from proton translocation across the membrane give rise to changes in interaction between dIII and dI alternately in the two halves of the molecule, and these drive the nucleotide-binding changes that are EXPERIMENTAL PROCEDURES. The rate of steady-state “reverse” transhydrogenation (see Reaction 1) in mixtures of dI and dIII was measured as the reduction of AcPdADϩ (an NADϩ analogue) by NADPH, from the absorbance change at 375 nm, as described previously [13]. The kinetic simulations of experimental data were carried out using the program GEPASI, version 3.21 [35]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
