Conformational gating of the dissimilatory sulfite reductase from Desulfovibrio vulgaris (Hildenborough). Synthesis, characterization, and stopped-flow kinetics studies of 1,5-IAEDANS-labeled desulfoviridin.

The siroheme prosthetic center in the dissimilatory sulfite reductase (desulfoviridin) from Desulfovibrio vulgaris (Hildenborough) readily binds exogenous ligands in the reduced state, but it does not do so in the oxidized state. In contrast, free oxidized siroheme in solution is observed to bind ligands rapidly. This can only be explained by a structural barrier that precludes ligand binding to the enzyme in the oxidized state but is removed after reduction. These observations suggest a redox-linked structural transformation that provides a gating mechanism for enzyme activation. The rate constants defining these structural perturbations, from oxidized-->reduced and reduced-->oxidized states, have been determined by monitoring changes in both the natural emission from desulfoviridin and the emission from a surface-bound fluorophore (1,5-IAEDANS). Consistent results were obtained from these two independent experimental measurements (at 25 degrees C: kox-->red approximately 8 s-1, kred-->ox approximately 0.05 s-1). Activation energies for each transition have been determined from Arrhenius plots (ox-->red: delta G* 16.5 kcal mol-1, delta H* 3.5 kcal mol-1, delta S* -43.8 cal K-1 mol-1; red-->ox: delta G* 19.2 kcal mol-1, delta H* 11.3 kcal mol-1, delta S* -26.6 cal K-1 mol-1). These data are used to further develop a functional model previously proposed for this class of enzyme [Lui, S. M., Soriano, A., & Cowan, J. A. (1993) J. Am. Chem. Soc. 115, 10483; Lui, S. M., Liang, W., Soriano, A., & Cowan, J. A. (1994) J. Am. Chem. Soc. 116, 4531].(ABSTRACT TRUNCATED AT 250 WORDS)