A high-affinity cbb3-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 O2 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 O2. 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 that 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 Mrs 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 cbb3-type subfamily of 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 cbb3-type oxidase. The Km values for O2 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 O2 delivery system. The derived Km value for O2 of the cbb3-type oxidase in membranes was 7 nM, which is six- to eightfold lower than that determined for the aerobic aa3-type cytochrome c oxidase. We conclude that the cbb3-type oxidase supports microaerobic respiration in endosymbiotic bacteroids.
Preisig
O
O
Mikrobiologisches Institut, Eidgenössische Technische Hochschule, Zürich, Switzerland.
Zufferey
R
R
Thöny-Meyer
L
L
Appleby
C A
CA
Hennecke
H
H
D000332:Aerobiosis; D000595:Amino Acid Sequence; D000693:Anaerobiosis; D001426:Bacterial Proteins / Q000032:analysis; D003573:Cytochrome b Group / Q000032:analysis; D003574:Cytochrome c Group / Q000032:analysis / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000032:analysis; D008566:Membranes / Q000378:metabolism; D008969:Molecular Sequence Data; D010101:Oxygen Consumption; D012230:Rhizobiaceae / Q000201:enzymology*; D017421:Sequence Analysis; D013053:Spectrophotometry; D013559:Symbiosis / Q000502:physiology*; D013772:Tetramethylphenylenediamine / Q000378:metabolism
D001426:Bacterial Proteins; D003573:Cytochrome b Group; D003574:Cytochrome c Group; C087009:FixN protein, bacteria; C080744:FixQ protein, Bacteria; D008565:Membrane Proteins; C080743:cytochrome FixO; C080742:cytochrome FixP; D003576:Electron Transport Complex IV; D013772:Tetramethylphenylenediamine