ENZYMATIC REDUCTION OF INORGANIC ANIONS - VARIABLE-TEMPERATURE STEADY-STATE AND PRE-STEADY-STATE KINETICS EXPERIMENTS TO MAP THE ENERGY PROFILE OF AN ENZYMATIC MULTIELECTRON REDOX REACTION - APPLICATION TO THE DISSIMILATORY SULFITE REDUCTASE FROM DESULFOVIBRIO-VULGARIS (HILDENBOROUGH)

Variable-temperature steady-state and pre-steady-state kinetics experiments have been carried out on the dissimilatory sulfite reductase (desulfoviridin) from Desulfovibrio vulgaris (Hildenborough). Activation free energies for reductive bond cleavage (Delta G(r)*) in SO32-, NO2-, NO, and NH2OH substrates have been evaluated from variable-temperature pre-steady-state kinetics data. Also, ground-state (Delta G(d)) and transition-state (Delta G*) contributions to the overall activation free energy (Delta G*) have been determined from steady-state experiments. The choice of siroheme cofactor for this class of enzyme most likely reflects two factors underlying a preference for pi-acceptor ligands. First, strong binding of substrate and weaker binding of product is promoted by the dominance of pi n-back-bonding. Second, population of an antibonding orbital in pi-acceptor substrates lowers the transition-state contribution to the activation free energy and serves to weaken the chemical bond that is to be reductively cleaved. These conclusions are supported by quantitative evaluation of activation barriers for substrates and reaction intermediates.