Use of stopped-flow spectrophotometry to establish midpoint potentials for redox proteins

Stopped-flow spectrophotometry was examined as a tool to assign midpoint potentials to protein redox half-reactions. The method involves the rapid mixing of protein and electron transfer mediator solutions and the determination of the absorbance of at least one of the reacting species or products at equilibrium. The utility of the method was demonstrated with two different redox proteins (nitrogenase iron protein and cytochrome c) with very different midpoint potentials. The overall errors ranged from about +/-0.5 to 3 mV. Advantages of the method include short times required for the experiments, the precision and accuracy of the method in comparison to other methods, the conservative use of valuable protein in the experiments and the ease of obtaining midpoint potentials for redox protein half-reactions, and the potential range covered by a single electron transfer mediator when the method involves mediated electron transfer. It is concluded that the stopped-flow spectrophotometry should be considered the method of choice for determining protein midpoint potentials.