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 leg ume root nodules by the use of a specialized respiratory electron tran sport chain terminating with an oxidase that ought to have a high affi nity for O-2. Previously, we suggested that the microaerobically and a naerobically induced fixNOQP operon of Bradyrhizobium japonicum might code for such a special oxidase, Here we report the biochemical charac teristics of this terminal oxidase after a 27-fold enrichment from mem branes of anaerobically grown B. japonicum wild-type cells, The purifi ed oxidase has TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) oxidase activity as well as cytochrome c oxidase activity. N-terminal amino a cid sequencing of its major constituent subunits confirmed the presenc e of the fixN, fixO, and fixP gene products. FixN is a highly hydropho bic, heme B-binding protein, FixO and FixP are membrane-anchored c-typ e cytochromes (apparent M(r)s of 29,000 and 31,000, respectively), as shown by their peroxidase activities in sodium dodecyl sulfate-polyacr ylamide gels. All oxidase properties are diagnostic for it to be a mem ber of the cbb(3)-type subfamily of the heme-copper oxidases. The FixP protein was immunologically detectable in membranes isolated from roo t nodule bacteroids, and 85% of the total cytochrome c oxidase activit y in bacteroid membranes was contributed by the cbb(3)-type oxidase. T he K-m values for O-2 of the purified enzyme and of membranes from dif ferent 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 respira tion in endosymbiotic bacteroids.