Evidence for dimer structure of proton-pumping cytochrome c oxidase, an analysis by radiation inactivation.

Abstract

Cytochrome c oxidases, purified from bovine heart and the thermophilic bacterium PS3, were irradiated with a high‐energy electron beam. The proton transport activities of both preparations and their electron transfer activities decreased as single exponential functions of the radiation dosage. Applying the target theory with alkaline phosphatase as an internal standard, the following functional molecular weights were obtained for cytochrome c oxidation and H+ pumping: 63‐73 kd and 160‐220 kd, respectively, for the bovine enzyme, and 80‐100 kd and 190‐230 kd for the PS3 enzyme. The results suggest that a dimer structure is necessary for H+ pumping, while a core part of monomer (presumably the largest two subunits, i.e. subunits I and II) is sufficient for cytochrome c oxidation.

Purification and characterization of two‐subunit cytochrome aa3 from Bacillus cereus

“…4) revealed a monomer (apparent molecular mass of 85 kDa). Interestingly, the elution pattern of our preparation (apparent molecular mass of 170kD) did not change as a result of lowering ionic strength (5O-5mM KCI) or changing to another detergent, as seems to occur with the bovine (Finel, 1987) and the PS3 enzyme (Sone and Kosako, 1986).…”

“…6; Table 2). This is also the case for cytochrome c oxidases from PS3 (Nicholls and Sone, 1984;Sone and Yanagita, 1982;Sone and Kosako, 1986), Thermus thermophilus , and B. subtilis (De Vrij and Konings, 1987). Different behaviour v^as observed in the Paracoccus denitrificans enzyme (Nalecz et ai, 1985) and the eukaryotic oxidase (NaJ'ecz et ai, 1983), in which a monomeric or dimeric state has been related to monophasic or biphasic kinetic behaviour, respectively.…”

“…Hydrodynamic characterizations of previously purified bacterial (Sone and Yanagita, 1982;Sone and Kosako, 1986) or bovine heart (Finel, 1987;Naleczefa/., 1983) aaa enzymes have suggested a dimeric structure, as assessed by gel filtration or sucrose density gradient centrifugation. Analysis of our purified preparation by gel filtration suggested a dimeric association (apparent molecular mass of 170 kDa) (Fig.…”

Cytochrome c oxidase in prokaryotes

“…A radiation inactivation analysis of the enzyme [83] argues strongly for a dimeric structure of the complex active in proton translocation in membranes, giving a size of 190-230 kDa, whereas determination for a complex functional in electron transport yields a mass of 80-100 kDa. Earlier results [76] showing a dimeric structure of the enzyme in the detergent lauroyl sarcosinate should be viewed with caution, since detergent binding was not taken into account.…”

The cytochrome oxidase subunit I and subunit III genes in Oenothera mitochondria are transcribed from identical promoter sequences

“…The unusually high histidine content of this polypeptide is also maintained in higher plants, with the 13 histidine residues present in identical positions in all COX IH subunits sequenced today. The conservation of membrane integration, the cytochrome c binding and the activity of proton translocation of the dimer COX (Sone and Kosako, 1986) are reflected in the relative positions of hydrophobic and hydrophilic regions seen in the hydropathy plots obtained for the COX III polypeptides of different species (not shown). The highly conserved sequence of unknown function near the amino terminus from positions 14 to 18, likely to form a $-turn (Chou and Fasman, 1978;Browning and RajBhandary, 1982), is also found in the higher plant polypeptide.…”