It has been a long-standing hypothesis that the endosymbiotic rhizobia (bacteroids) cope with a concentration of 10 to 20 nM free O 2 in legume root nodules by the use of a specialized respiratory electron transport chain terminating with an oxidase that ought to have a high affinity for O 2 . Previously, we suggested that the microaerobically and anaerobically induced fixNOQP operon of Bradyrhizobium japonicum might code for such a special oxidase. Here we report the biochemical characteristics of this terminal oxidase after a 27-fold enrichment from membranes of anaerobically grown B. japonicum wild-type cells. The purified oxidase has TMPD (N,N,N,N-tetramethyl-p-phenylenediamine) oxidase activity as well as cytochrome c oxidase activity. N-terminal amino acid sequencing of its major constituent subunits confirmed the presence of the fixN, fixO, and fixP gene products. FixN is a highly hydrophobic, heme B-binding protein. FixO and FixP are membraneanchored c-type cytochromes (apparent M r s of 29,000 and 31,000, respectively), as shown by their peroxidase activities in sodium dodecyl sulfate-polyacrylamide gels. All oxidase properties are diagnostic for it to be a member of the cbb 3 -type subfamily of the heme-copper oxidases. The FixP protein was immunologically detectable in membranes isolated from root nodule bacteroids, and 85% of the total cytochrome c oxidase activity in bacteroid membranes was contributed by the cbb 3 -type oxidase. The K m values for O 2 of the purified enzyme and of membranes from different B. japonicum wild-type and mutant strains were determined by a spectrophotometric method with oxygenated soybean leghemoglobin as the sole O 2 delivery system. The derived K m value for O 2 of the cbb 3 -type oxidase in membranes was 7 nM, which is six-to eightfold lower than that determined for the aerobic aa 3 -type cytochrome c oxidase. We conclude that the cbb 3 -type oxidase supports microaerobic respiration in endosymbiotic bacteroids.

We report the discovery of a Bradyrhizobium

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.