Preparation of a one-subunit cytochrome oxidase from Paracoccus denitrificans: spectral analysis and enzymic activity

Abstract

Cytochrome c oxidase was isolated from Paracoccus denitrificans as a two-subunit enzyme. Chymotrypsin-catalyzed proteolysis reduced the molecular weight of each subunit by about 8000. The spectral properties of this preparation, as well as its Km for cytochrome c(1.7 muM), remained unchanged with respect to the native enzyme. Vmax was reduced by about 55% when assayed in Triton X-100 or in Triton X-100 supplemented with asolectin. Following further proteolysis by Staphylococcus aureus V8 protease, subunit I remained unchanged as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas subunit II was split into small peptides. These were removed by ion-exchange high-performance liquid chromatography. The one-subunit enzyme had an apparent molecular weight of 43,000. The reduction of molecular weight was also confirmed by the diminution of the ultraviolet/Soret absorption ratio. This value was 1.8-2.1 for the native enzyme and 1.3-1.5 for the one-subunit enzyme. The spectral properties (including the spectrum CO reduced minus reduced) were not modified by the proteolytic treatment, indicating that cytochromes a and a3 were present in equal amounts. The lack of spectral alteration and the known close association of the copper B atom with cytochrome a3 suggest that copper B is also contained within the one-subunit enzyme. The Km of the one-subunit oxidase was similar to that of the two-subunit enzyme; Vmax was decreased by about 50%. The activity of the one-subunit oxidase had a salt-dependent maximum at 30 mM KCl, almost identical with that of the undigested enzyme, and was inhibited by micromolar concentrations of KCN.

Methionine-393 is an axial ligand of the heme b558 component of the cytochrome bd ubiquinol oxidase from Escherichia coli

“…= 6, g: = 2, and the rhombic signal at gy,z = 5.5, 6.3 assigned to heme £>595 in wild-type oxidase (Meinhardt et al, 1989;Muller et al, 1988). The spectrum shows no evidence of the g = 3.3 signal previously assigned to heme £?558 (Meinhardt et al, 1989;Muller et al, 1988). The high-spin form of heme b$$z in this mutant appears to be adding to the intensity of the axial g = 6 signal, but this was not quantified.…”

“…The spectrum shows a high-spin axial signal at gX:}. = 6, g: = 2, and the rhombic signal at gy,z = 5.5, 6.3 assigned to heme £>595 in wild-type oxidase (Meinhardt et al, 1989;Muller et al, 1988). The spectrum shows no evidence of the g = 3.3 signal previously assigned to heme £?558 (Meinhardt et al, 1989;Muller et al, 1988).…”

“…Heme £7595 is a high-spin heme center (Hata et al, 1985;Hata-Tanaka et al, 1987;Meinhardt et al, 1989; Rothery & Ingledew, 1989). Heme d is a chlorin (Sotiriou & Chang, 1988;Timkovich et al, 1985;Vavra et al, 1986) and has also been shown to be in a high-spin state (Meinhardt et al, 1989;Muller et al, 1988). A small percentage of this heme d is also found in a low-spin form; however, this has been proposed to be an artifact, possibly caused by freeze/thaw treatment.…”

Structural features of cytochrome oxidase

“…The major subunit of cytochrome oxidase carries both haem A groups and contains the bimetallic oxygen-binding site (Holm et al 1987;Muller et al 1988;Haltia et al 1989.). As Fig.…”

Effect of chemical modification of lysine amino groups on redox and protonmotive activity of bovine heart cytochrome c oxidase reconstituted in phospholipid membranes

“…In mitochondria and certain bacteria, including Paracoccus denitrificans, aerobic oxidation of ferrocytochrome c by the oxidase is also associated with proton release (Wikstrom, 1976;Wikstrom et al, 1985;Papa, 1988). This process is generally considered to represent uphill proton translocation (H+ pumping) from the inner to the outer aqueous phase, its mechanism remaining, however, to be established (Wikstrom et al, 1985;Mitchell et al, 1985;Mitchell, 1987; Malmstróm, Subunit I may perform basic processes of redox catalysis (Buse et al, 1987;Müller et al, 1988) and protonmotive activity (Hon-nami & Hoshima, 1984). Subunit II mediates reaction of cytochrome c with the oxidase (Capaldi et al, 1982).…”