Purification and partial characterization of the membrane-bound haem-containing proteins from Acinetobacter calcoaceticus LMD 79.41

Abstract

Cytoplasmic membranes of Acinetobucter cdcoaceticus cells, cultured under acetate-limiting conditions, contain cytochrome bSs4 and a cytochrome o-containing oxidase. Both have been purified to homogeneity and characterized. Cytochrome bss4 is a monomeric protein (molecular mass 70 kDa) with a midpoint potential of + lo0 mV (in the membrane it has most probably a value of +50 mV). The cytochrome o-containing oxidase seems to be an aPy8 protein since the molecular mass of the native protein was estimated to be 150 kDa and the molecular masses of the subunits, determined by SDS-polyacrylamide-gel electrophoresis, are 55, 41, 33 and 17 kDa. Redox spectroscopy of the purified complex shows the presence of a cytochrome bss5,563 having a midpoint potential of approximately + 160 mV (both in purified form and in the membrane). CO difierence spectroscopy shows the presence of a second b-type cytochrome, viz. cytochrome 0. Cytoplasmic membranes of A. calcoaceticus cells grown under oxygen-limiting conditions also show the presence of the cytochrome bSs4 and the cytochrome o-containing oxidase. In addition a protein has been solubilized with the spectral characteristics of a cytmhrome d-containing oxidase. The cytochrome o-and d-containing oxidases appear to be similar to those reported for Esckrichia coli and Profeus mirabilis. On the other hand, cytochrome bSs4 has no counterpart in these organisms since the cytochrome bSs6 described for E. cofi is quite dissimilar.

Modified, large-scale purification of the cytochrome o complex (bo-type oxidase) of Escherichia coli yields a two heme/one copper terminal oxidase with high specific activity

“…(1) The enzyme contains four subunits, as do cytochrome o preparations from other organisms (Dokter et al, 1990;Georgiou & Webster, 1987;Matsushita et al, 1987;Schrattenholz et al, 1989;Sone et al, 1990a) as well as the similar ao3-type cytochrome c oxidase from Bacillus PS3 (Sone et F and G to the purified cytochrome o complex in detergent. Cytochrome o complex (5 ng) was incubated with the antibody in 200 nL of PBS overnight at 5 °C.…”