A high-affinity cbb3-type cytochrome oxidase terminates the symbiosis-specific respiratory chain of Bradyrhizobium japonicum

Abstract

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.

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

“…It belongs to the so-called super-family of heme-copper oxidases [3] and is encoded by the fixNOQP operon [1]. 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].…”

“…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].…”

“…FixN is an integral membrane protein with up to 14 putative transmembrane a-helices [1,2]. It contains a low-and a highspin heine B cofactor, the latter being associated with copper (CUB) in a binuclear center that is the catalytic site of oxygen reduction [3,4,6,7].…”

A decade of crystallization drops: Crystallization of the cbb3 cytochrome c oxidase from Pseudomonas stutzeri

“…Surprisingly, the iron compounds in general favored the formation of the orthorhombic crystals used for structure determination. While the addition of 16 mM K 3 Fe(CN) 6 or K 4 Fe(CN) 6 reproducibly induced crystal growth, this process was impaired at 8 mM and abolished in their absence. This finding can be correlated with the binding of [Fe(CN) 6 ] 32 inside a positively charged cavity formed by the periplasmic CcoP domains of the four cbb 3 -CcO complexes in the asymmetric unit [ Fig.…”

“…Interestingly, ctype cytochromes from horse heart, P. aeruginosa or Pichia pastoris could not replace the S. cerevisiae protein which suggests a transient but specific contact. K 3 Fe(CN) 6 or K 4 Fe(CN) 6 were initially applied to crystallize cbb 3 -CcO either in a fully oxidized or reduced state. Surprisingly, the iron compounds in general favored the formation of the orthorhombic crystals used for structure determination.…”