Site-specific antibodies against hydrophilic domains of subunit III of bovine heart cytochrome c oxidase affect enzyme function.

Antibodies were raised against conserved amino acid sequences in four extramembranous portions of subunit III (sIII) from beef cytochrome c oxidase (COX) and the role of these domains in the functional activities of the enzyme was investigated. The binding of one antipeptide antibody corresponding to an externally exposed (facing the intermembrane space) domain of COX sIII (amino acids 180-189 in the primary sequence) exhibited a 30-50% stimulation of electron transfer activity in both detergent-dispersed COX and COX incorporated into phospholipid vesicles (COV). Antibody binding to two different matrix-faced domains (amino acids 57-66 and 148-159 in the sequence) resulted in small stimulations (10-25%) of COX electron transfer activity. The remaining antipeptide antibody (against amino acids 119-128) had no effect on electron transfer activity of COX in detergent solution, but exhibited a slight inhibition of activity (15%) in COV. The mechanism of antibody-induced stimulation of COX electron transfer activity was determined to be an increase in the maximum velocity of the enzyme and not due to a change in the apparent K(m) of cytochrome c interaction with COX as determined by steady state kinetic assays. Antibody binding to COX in COV increased the respiratory control ratio (an indicator of endogenous proton permeability) of COV, but had no effect on the vectorial proton pumping activity of COV. These results suggest that these conserved, hydrophilic domains of COX sIII are conformationally linked to the electron transfer function of the enzyme in subunits I and II and that sIII may serve as a regulatory subunit for COX electron transfer and proton pumping activities.