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

Author:

Preisig O1,Zufferey R1,Thöny-Meyer L1,Appleby C A1,Hennecke H1

Affiliation:

1. Mikrobiologisches Institut, Eidgenössische Technische Hochschule, Zürich, Switzerland.

Abstract

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.

Publisher

American Society for Microbiology

Subject

Molecular Biology,Microbiology

Reference40 articles.

1. Leghemoglobin and Rhizobium respiration. Annu;Appleby C. A.;Rev. Plant Physiol.,1984

2. Cytochromes of Rhizobium;Appleby C. A.;Nature (London),1958

3. Appleby C. A. and F. J. Bergersen. 1980. Preparation and experimental use of leghaemoglobin p. 315-335. In F. J. Bergersen (ed.) Methods of evaluating biological nitrogen fixation. John Wiley & Sons Chichester United Kingdom.

4. Effects of concentrations of substrates supplied to N2-fixing soybean bacteroids in flow chamber reactions;Bergersen F. J.;Proc. R. Soc. Lond. Ser. B,1993

5. Genetic analysis of the cytochrome c-aa3 branch of the Bradyrhizobium japonicum respiratory chain;Bott M.;Mol. Microbiol.,1990

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