ABSTRACT
Under fully reduced conditions, reassociation kinetics of CO were studied in several terminal oxidases containing copper in their binuclear center. The purified Paracoccus denitrificans ba3-type quinol oxidase was found to recombine with CO monophasically (tau 25-30 ms) like oxidases of the bo type from Escherichia coli, the caa3 type from Bacillus halodurans FTU, and the bo type from Methylobacillus flagellatum KT. Oxidase of the aa3 type from bovine heart recombined with CO monophasically at a higher rate (tau 16-19 ms) than the studied copper-containing bacterial oxidases. After prolonged incubation in the presence of CO, oxidases of the ba3 and aa3 types changed their CO-binding properties. The contribution of the slow component was diminished while new fast components arose. Measurement of the metal content in the oxidases indicated that during the incubation, the enzymes lost their copper, the process being accompanied by the appearance of a fast CO recombination rate resembling that of the non-copper oxidases of the bd type from E. coli and the bb type from Bacillus halodurans FTU. This points to a role of copper in CO binding by terminal oxidases.
Subject(s)
Carbon Monoxide/metabolism , Copper/physiology , Electron Transport Chain Complex Proteins , Electron Transport Complex IV/metabolism , Escherichia coli Proteins , Oxidoreductases/metabolism , Animals , Cattle , Cytochrome b Group/metabolism , Cytochromes/metabolismABSTRACT
Expression of the quinol oxidase from Paracoccus denitrificans has been examined using a polyclonal antibody directed against subunit II and a promoter probe vector carrying the promoter region of the qox operon. Under aerobic conditions nitrate and nitrite act as specific inducers of the expression. To obtain an enzymatically competent quinol oxidase complex, an intact ctaB gene is required, which constitutes part of the cta operon coding for the aa3 cytochrome c oxidase of P. denitrificans. Deletion of ctaB leads to a change in heme composition of the quinol oxidase with heme b replacing the high-spin heme a of the binuclear center, causing loss of electron transport activity.
Subject(s)
Cytochrome b Group/metabolism , Electron Transport Complex IV/metabolism , Paracoccus denitrificans/enzymology , Antibodies/metabolism , Base Sequence , Blotting, Western , Circular Dichroism , Cytochrome b Group/genetics , Electron Transport Complex IV/genetics , Gene Expression Regulation, Bacterial , Gene Expression Regulation, Enzymologic , Heme/metabolism , Magnetics , Molecular Sequence Data , Nitrates/metabolism , Nitrites/metabolism , Operon , Paracoccus denitrificans/genetics , Peptide Fragments/immunology , Protein Conformation , Spectrophotometry, AtomicABSTRACT
The ba3 quinol oxidase from Paracoccus denitrificans has been purified by a new protocol leading to significantly higher yields than previously reported (Richter et al. (1994) J. Biol. Chem. 269, 23079-23086). In an SDS PAG an additional protein band compared with the previous preparation appears, which can be identified as the major form of subunit II. All protein bands can be assigned to genes of the qox operon by N-terminal sequencing, indicating that the oxidase consists of four subunits. In addition to one heme A, one heme B, and one copper atom, the preparation contains two ubiquinone molecules per enzyme. The oxidase is further characterized by electron paramagnetic resonance (EPR), circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopy.
