Expression of the quinol oxidase from Paracoccus denitrijicans has been examined using a polyclonal antibody directed against subunit I1 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 eta operon coding for the aa, 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.Keywords : cytochrome-c oxidase ; high-spin heme ; CtaB ; nitratehitrite regulation ; NarL. [2] proposed a different heme composition. When they analyzed the heme composition of membranes isolated from a strain deleted in both copies of the aa, subunit I genes they concluded that both heme sites of the remaining quinol oxidase are occupied by b-type hemes. This discrepancy in the reported heme composition was explained by a different genetic background of the respective Paracoccus strains [2].In this report we present expression studies of the quinol oxidase in different strains as well as the characterization of the purified enzymes from these strains. The heme composition of the quinol oxidase is changed by deletion of the cta operon as can be seen from visible absorption and magnetic circular dichroism (MCD) spectroscopic analysis. MATERIALS AND METHODSPlasmids, bacteria and growth condition. Paracoccus denitri,fcans strains Pd1222 [ 181, (3440 (deletion of the f l c operon coding for the cytochrome be, complex, dfbc::neo) [19], ST4 (lacking the complete eta operon, dcta::neo) and ST4P (ST4 complemented with ctaC and ctaB in trans) [14] were grown aerobically on succinate medium, which was supplemented with KNO, or KNO, where indicated. P: denitrificans cells were harvested in the logarithmic growth phase, disrupted and membranes isolated as described previously [20]. Anaerobic cultures were grown in stoppered flasks with a paraffin oil overlay. E. coli cells were cultured in TY medium (0.8 % tryptone, 0.5 % yeast extract, 0.5% NaCI, pH 7.3), treated with lysozyme, and subsequently disrupted by sonication.The cytochrome ba, promoter region was amplified from an EcoRI fragment of the qox operon (position -339 to +257) cloned into pUC18 (reverse primer and primer MU2: 5'-TCTA-GATGTACATCATTGGACTACCCG-?) as was the genetically engineered E. COli P-galactosidase gene (universal primer and primer MG 1 : S-TTGGTACCAATGTACAAGGTTTTATCCGCorrespondence to 0.

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.