Analysis Of The Structural And Functional Role Of The Dicyclohexylcarbodiimide (Dccd) Binding Site (E90-H212-Y246) In Subunit Iii Of Bovine Heart And Rhodobacter Sphaeroides Cytochrome C Oxidase Using Mutagenesis And Chemical Modification

Subunit III (SIII) of cytochrome c oxidase (COX) is conserved in most prokaryotes and eukaryotes. A triad of amino acids in SIII (E90, H212, Y246) is also conserved across most species; however, the role of these critical amino acids in oxidase structure and function is unresolved. This triad of amino acids in SIII is buried within the membrane bilayer and each residue resides on a separate α-helix. Chemical modification of E90 in SIII with DCCD blocks proton translocation in bovine heart oxidase. DCCD binding induces a conformational change in SIII to make it more labile to limited proteolysis. DCCD also causes suicide inactivation in both the bovine and Rhodobacter sphaeroides enzymes. Mutagenesis of the conserved triad resides in Rhodobacter results in COX enzymes with altered activities. Both H212F and H212A mutants in SIII induce suicide inactivation and inhibit proton pumping, while retaining wild type SIII content. Alternatively, E90A results in a purified enzyme with dramatically decreased SIII content which exhibits suicide inactivation and reduced proton pumping activity similar to the enzyme depleted in SIII. In contrast, Y246F shows wild-type electron transfer and proton pumping activities as well as wild-type SIII content. The E90H-H212E double mutant has wild-type activities, whereas E90H exhibits decreased electron transfer and lowered proton pumping activities. Finally, the H212E mutant is not expressed in the bacteria suggesting either an assembly or protein folding problem. These results taken together suggest that the E90-H212 linkage (either a hydrogen bond or electrostatic interaction) in SIII is critical for optimal cytochrome c oxidase function. Disrupting this interaction induces deleterious effects on proton pumping efficiency and induces suicide inactivation. This disruption can lead to rearrangement of phospholipids intimately associated with SIII. We propose that the E90-H212 linkage may be involved in conformational changes in SIII that when translated to subunit I during enzyme turnover results in optimal enzyme function.