ASSEMBLY AND FUNCTION OF THE CYTOCHROME CBB(3) OXIDASE SUBUNITS IN BRADYRHIZOBIUM-JAPONICUM

The Bradyrhizobium japonicum cbb(3)-type cytochrome oxidase, which sup ports microaerobic respiration, is a multisubunit enzyme encoded by th e genes of the fixNOQP operon, We investigated the contribution of the individual subunits to function and assembly of the membrane-bound co mplex, In-frame deletion mutants of fixN, fixO, and fixQ, and an inser tion mutant of fixP were constructed. All mutants, except the fixQ mut ant, showed clearly altered absorption difference spectra of their mem branes and decreased oxidase activities, and they were unable to fix n itrogen symbiotically, The presence of the individual subunits was ass ayed by Western blot analysis, using subunit-specific antibodies, and by heme staining of the c-type cytochromes FixO and FixP. These analys es led to the following conclusions: (i) FixN and FixO are necessary f or assembly of the multimeric oxidase, (ii) FixN and FixO assemble ind ependently of FixP, and (iii) FixQ is not required for complex formati on and, therefore, does not seem to be an essential subunit, The possi ble oxidase biogenesis pathway involves the formation of a primary cor e complex consisting of FixN and FixO, which allows the subsequent ass ociation with FixP to form the complete enzyme.