title: Characterization of an fdxN mutant of Rhodobacter capsulatus indicates that ferredoxin I serves as electron donor to nitrogenase.
abstract: A mutant of Rhodobacter capsulatus, carrying an insertion into the fdxN gene encoding ferredoxin I (FdI), has been studied by biochemical analysis and genetic complementation experiments. When compared to the wild-type strain, the fdxN mutant exhibited altered nitrogen fixing ability and 20-fold lower levels of nitrogenase activity as assayed in vivo. When assayed in vitro with an artificial reductant, nitrogenase activity was only 3- to 4-fold lower than in the wild type. These results suggested that the FdI-deleted mutant had impaired electron transport to nitrogenase. Immunochemical assay of both nitrogenase components showed that the fdxN mutant contained about 4-fold less enzyme than wild-type cells. Results of pulse-chase labeling experiments using [35S]methionine indicated that nitrogenase was significantly less stable in the FdI-deleted mutant. When a copy of fdxN was introduced in the mutant in trans, the resulting strain appeared to be fully complemented with respect to both diazotrophic growth and nitrogenase activity. Depending on whether fdxN expression was driven by a nif promoter or a fructose-inducible promoter, FdI was synthesized either at wild-type level or in 10-fold lower amounts. The strain producing 10-fold less FdI did, however, display normal N2-fixing ability. Analysis of cytosolic proteins by bidimensional electrophoresis revealed that the fdxN mutant produced a 14 kDa polypeptide in amounts about 3-fold greater than wild-type cells. This protein was identified by N-terminal microsequencing as a recently purified [2Fe-2S] ferredoxin, called FdV, which cannot reduce nitrogenase. It is concluded that FdI serves as the main electron donor to nitrogenase in R. capsulatus and that an ancillary electron carrier, distinct of FdV, is responsible for the residual nitrogenase activity observed in the FdI-deleted mutant.
mesh_terms: D001483:Base Sequence; D004579:Electron Transport; D004795:Enzyme Stability; D005288:Ferredoxins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009591:Nitrogenase / Q000378:metabolism*; D010957:Plasmids; D016354:Rhodobacter capsulatus / Q000235:genetics / Q000378:metabolism*
chemical_list: D005288:Ferredoxins; D009591:Nitrogenase