Conformational changes in the membrane-bound hydrogenase of Bradyrhizobium japonicum. Evidence that the redox state of the enzyme affects its accessibility to protease and membrane-impermeant reagents.
The sensitivity of the membrane-bound hydrogenase of Bradyrhizobium japonicum to inactivation by proteases and membrane-impermeant protein modification reagents was compared under hydrogen versus oxygen. In membrane vesicles, the half-life of enzyme inactivation by trypsin of the H2-reduced enzyme was approximately 10 min, whereas O2-oxidized enzyme was much less sensitive to trypsin inactivation (half-life of over 90 min). Diazobenzene sulfonate (DABS) affected the enzyme activity in a manner similar to proteases. With DABS, the enzyme had a half-life of 2-3 min under H2 versus over 30 min under O2. Experiments in which the gas phase (containing either H2 or O2) available to the membranes was changed prior to the protease or chemical modification treatments indicated that it is the redox state of the enzyme at the time of the treatment which determines the sensitivity of the enzyme to inactivation. The redox-dependent differences in the behavior of the membrane-bound enzyme were attributed to changes in the accessibility of the small (33 kDa) subunit. The kinetics of enzyme inactivation by trypsin, under H2, correlated very well with the degradation of the intact 33-kDa subunit, whereas the large subunit (65 kDa) was rather resistant to proteolytic degradation. DABS treatment was found to decrease the reactivity of the small subunit to its antibody concomitant with enzyme inactivation under H2, but without such an effect on the O2-oxidized enzyme. In contrast to the results with the membrane-bound enzyme, purified dehydrogenase was found to be equally susceptible to inactivation by proteolysis or chemical modification irrespective of whether the treatments were performed under H2 or O2. These results indicate that, in the membrane, hydrogenase undergoes a redox-linked conformational change, whereby the small subunit of the enzyme becomes more accessible to external reagents when the enzyme is in its reduced form.
D002462:Cell Membrane / Q000201:enzymology; D003979:Diazonium Compounds / Q000494:pharmacology; D006864:Hydrogenase / Q000378:metabolism*; D007202:Indicators and Reagents; D007700:Kinetics; D010084:Oxidation-Reduction; D010447:Peptide Hydrolases; D011487:Protein Conformation; D012231:Rhizobium / Q000201:enzymology*; D013425:Sulfanilic Acids / Q000494:pharmacology
D003979:Diazonium Compounds; D007202:Indicators and Reagents; D013425:Sulfanilic Acids; D006864:Hydrogenase; D010447:Peptide Hydrolases; C000469:diazobenzenesulfonic acid