Assembly and Function of the Cytochrome cbb Oxidase Subunits in Bradyrhizobium japonicum

Abstract

The Bradyrhizobium japonicum cbb 3 -type cytochrome oxidase, which supports microaerobic respiration, is a multisubunit enzyme encoded by the genes of the fix-NOQP operon. We investigated the contribution of the individual subunits to function and assembly of the membrane-bound complex. In-frame deletion mutants of fixN, fixO, and fixQ, and an insertion mutant of fixP were constructed. All mutants, except the fixQ mutant, showed clearly altered absorption difference spectra of their membranes and decreased oxidase activities, and they were unable to fix nitrogen symbiotically. The presence of the individual subunits was assayed by Western blot analysis, using subunit-specific antibodies, and by heme staining of the c-type cytochromes FixO and FixP. These analyses led to the following conclusions: (i) FixN and FixO are necessary for assembly of the multimeric oxidase, (ii) FixN and FixO assemble independently of FixP, and (iii) FixQ is not required for complex formation and, therefore, does not seem to be an essential subunit. The possible oxidase biogenesis pathway involves the formation of a primary core complex consisting of FixN and FixO, which allows the subsequent association with FixP to form the complete enzyme.

Histidine 131, not histidine 43, of the Bradyrhizobium japonicum FixN protein is exposed towards the periplasm and essential for the function of the cbb3‐type cytochrome oxidase

“…The mutations were verified by sequencing. The corresponding B. japonicum mutants were generated by cointegration of pSUP202 derivatives into the chromosome of a AfixNOQP strain (Bj4503) as described previously [5].…”

“…For the fixN'-'phoA fusion, the phoA gene of pCH39X (pCH39 [21], with a XbaI linker in the XhoI site) was cloned into the Pstl site of pRJ4598, resulting in pRJ4604. The plasmids pRJ4598 and pRJ4604 were cointegrated into the chromosome of B. japonicum Bj4503 (AfixNOQP) as described in [5]. The correct cointegrates were confirmed by Southern blot hybridizations.…”

“…Tetramethyl-p-phenylenediamine (TMPD) oxidase activity, cytochrome c oxidase activity with reduced horse heart cytochrome c as an electron donor and the nitrogen fixation activity were measured as described before [5].…”

“…Membrane fractions were isolated as described elsewhere [5]. Determination of protein concentrations, SDS-PAGE according to Laemmli [24], heine stains and Western blot analyses with antibodies against FixN, FixO, FixP and cytochrome cl were performed as described previously [5].…”

“…The purified oxidase contains at least three subunits [4]. The special structural feature of the cbb3-type oxidase is the association of a subunit I (FixN protein) with two c-type cytochromes, FixO and FixP, representing subunit II and subunit III, respectively, which are anchored in the cytoplasmic membrane by their hydrophobic N-termini and which are exposed to the periplasm [1,2,4,5]. FixN is an integral membrane protein with up to 14 putative transmembrane a-helices [1,2].…”