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

Abstract

Cytochrome c-oxidase type aa3 (EC 1.9.3.1) was purified to homogeneity from vegetative Bacillus cereus by ion-exchange and hydroxylapatite chromatography in the presence of Triton X-100. Gel filtration analysis suggested a dimeric structure apparently 172 kDa in size; however, only a monomer of 81 kDa was detected when analysed by non-denaturing gel electrophoresis. Denaturing gel electrophoresis analysis of the protein showed the presence of two subunits (51 and 30 kDa). Atomic absorption and visible spectroscopy showed typical aa3 redox centres with haem a iron and copper in a ratio of 22 nmol and 35 ng-atom per mg protein, respectively. No haem c was found associated with the purified enzyme in the conditions reported here. Oxidase activity was fully reconstituted by phospholipids in the presence of N,N,N',N'-tetramethyl-p-phenylenediamine or reduced yeast cytochrome c (but not horse cytochrome c) as electron donors. This activity was abolished by cyanide and carbon monoxide.

Two different aa3‐type cytochromes can be purified from the bacterium Bacillus cereus

“…One isoform bears a cytochrome c moiety in the smaller subunit, resembling the cyto-chrome caa3 described in some thermophilic Bacillus species (Ludwig, 1987). The second isoform, free of cytochrome c, appears to be identical to the aa3 complex that we previously described in vegetative B. cereus (Garcia-Horsman et al, 1991).…”

“…Pur$cation procedure Cytochrome aa3 was purified as described elsewhere (Garcia-Horsman et al, 1991). Cytochrome caa3 was purified as stated in Results.…”

“…LDSjPAGE was performed according to Laemmli (1970) at 4 T as described elsewhere (Garcia-Horsman et al, 1991). The SDSjPAGE method reported by Schagger and von Jagow (1987) was used as previously described (Garcia-Horsman et al, 1991).…”

“…In Bacillus firmus RAB a cytochrome c co-purifies with the oxidase (Kitada and Krulwich, 1984). Alternatively, in Bacillus cereus (Garcia-Horsman et al, 1991) as well in B. subtilis (De Vrij et al, 1986), a cytochrome ua3 has been purified and characterized which does not contain a heme c polypeptide. However, a caa3 complex has been described in B. subtilis .…”

Oxygen-dependent transcriptional regulation of cytochrome aa3 in Bradyrhizobium japonicum

“…The most obvious environmental change in the transition from fully aerobic cultured cells to bacteroids is the profound decrease in free 02 levels (4,6,8). Since free 02 levels are so low under symbiotic conditions, estimated at 11 nM (4), and since the cytochrome aa3 terminal oxidase has a relatively low 02 affinity in both B. japonicum (28) and other prokaryotic systems (9,11,37) (an apparent 02 affinity of 4 to 7 lLM), cytochrome aa3 may not be physiologically active at very low 02 concentrations, and transcription will thus be repressed.…”