Rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGR) is extremely sensitive to oxidative inactivation by low concentrations (micromolar) of glutathione disulfide (GSSG) even in the presence of millimolar concentrations of glutathione (GSH). Inactivation involves the formation of an intramolecular protein-SS-protein disulfide in thiol/disulfide redox equilibrium with the reduced, active enzyme (Cappel, R.E., and Gilbert, H.F. (1988) J. Biol. Chem. 263, 12204-12212). In the absence of dithiothreitol, HMGR oxidation has been previously shown to cross-link the microsomal enzyme into a covalent dimer (Ness, G.C., McCreery, M.J., Sample, C.E., Smith, M., and Pendelton, L.C. (1985) J. Biol. Chem. 260, 12391-12393). Examination of the extent of HMGR cross-linking and residual HMGR activity in microsomes equilibrated with glutathione redox buffers establishes that inactivation and cross-linking result from oxidation of different dithiol pairs. The thiol/disulfide oxidation potential (K(ox)) for the oxidative inactivation of HMGR, E(SH)2,active + GSSG<-->E(S-S)inactive + 2 GSH, is 0.67 +/- 0.07 M. However, the equilibrium constant for HMGR cross-linking, E(SH)2,monomer + GSS<-->E(S-S)dimer + 2 GSH, is 0.19 +/- 0.02 M, significantly lower than that for inactivation (p < 0.001). Because of the significantly different oxidation potentials and the lack of a linear relationship between cross-linking and inactivation, the two processes must involve two different sets of vicinal dithiols. HMGR becomes 5-10-fold more difficult to oxidize in the presence of saturating levels of the substrate, HMG-CoA. Both inactivation and cross-linking exhibit significantly lower oxidation potentials in the presence of this substrate, 0.072 +/- 0.01 and 0.047 +/- 0.007 M, respectively. The decrease in oxidation potential caused by substrate binding is observed for both inactivation and cross-linking, showing that both processes are affected by the binding of substrate to the enzyme. The dithiols involved in HMGR subunit cross-linking are 2-3-fold more difficult to oxidize than the dithiols that affect the enzyme activity. Thus, the observation of partial cross-linking of HMGR in vivo would imply that conditions are sufficiently oxidizing to result in significant enzyme inactivation. The extreme thermodynamic sensitivity of HMGR to oxidative inactivation and cross-linking in glutathione redox buffers that span the physiological redox state implies that thiol/disulfide redox state changes could provide a mechanism for regulating the activity and/or stability of this enzyme.