A high-affinity cbb(3)-type cytochrome oxidase terminates the symbiosis-specific respiratory chain of Bradyrhizobium japonicum

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 membrane-anchored 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.