PHYSICAL AND FUNCTIONAL-CHARACTERIZATION OF MONOMERIC AND DIMERIC EUKARYOTIC CYTOCHROME-C OXIDASES

Cytochrome c oxidases were isolated from heart tissue of beef (Bos tau ros), sheep (Ovis aries), horse (Equus caballus), pig (Sus scrofa) (na tive dimers) and hammerhead shark (Sphyrna lewinii) (native monomer). Limited proteolysis of dimeric enzymes selectively depleted subunit II I, resulting in monomerisation and a blue shift (2nm) of the reduced a lpha band to the same wavelength maximum (603nm) as that of the hammer head shark enzyme. Monomeric enzymes retain the ability to accept elec trons rapidly from cytochrome c, and the second-order rate constants f or electron transfer between cytochromes c and a are reported. The ste ady-state kinetics of both native and subunit III-depleted cytochrome c oxidases were biphasic, thus ruling out any explanation for this beh aviour that depends on cooperation between functional units (monomers) within a dimer. Functional integrity of the subunit III-depleted enzy me prepared by proteolysis was maintained during multiple turnover, in contrast to reports elsewhere of loss of activity caused by subunit I II removal by other means. A model is proposed to explain this differe nce, in which removal of a hydrophobic membrane-spanning segment of su bunit III leads to monomerisation but a residual extra-membrane segmen t is retained, preserving the functional integrity of the enzyme. Copy right (C) 1996 Elsevier Science Inc.