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 THEDISSIMILATORY SULFITE REDUCTASE FROM DESULFOVIBRIO-VULGARIS (HILDENBOROUGH)

Variable-temperature steady-state and pre-steady-state kinetics experi ments have been carried out on the dissimilatory sulfite reductase (de sulfoviridin) 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-temperatu re pre-steady-state kinetics data. Also, ground-state (Delta G(d)) and transition-state (Delta G) contributions to the overall activation f ree energy (Delta G) have been determined from steady-state experimen ts. The choice of siroheme cofactor for this class of enzyme most like ly reflects two factors underlying a preference for pi-acceptor ligand s. 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 transitio n-state contribution to the activation free energy and serves to weake n the chemical bond that is to be reductively cleaved. These conclusio ns are supported by quantitative evaluation of activation barriers for substrates and reaction intermediates.