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.

A new ferredoxin has been purified from the photosynthetic bacterium Rhodobacter capsulatus. It is the sixth ferredoxin to be isolated from this bacterium and it was called FdVI.Its primary structure was established based on amino acid sequence analysis of the protein and of peptides derived from it. It is composed of 106 residues including five cysteines. The calculated mass of the polypeptide is 11402.6 Da which matches the experimental value determined by electrospray mass spectrometry. Amino acid sequence comparison revealed that ferredoxin VI (FdVI) is strikingly similar to a ferredoxin from Caulobacter crescentus and to the putidaredoxin from Pseudomonas putida.FdVI exhibited an ultraviolet-visible absorption spectrum typical for a [2Fe-2S] ferredoxin. EPR spectroscopy of the reduced protein showed a nearly axial signal similar to that of mitochondria1 and €? putida ferredoxins.FdVI is biosynthesized in cells growing anaerobically under either nitrogen-sufficient or nitrogen-deficient conditions. Although the function of FdVI is unknown, its structural resemblance to [2Fe-2S] ferredoxins known to transfer electrons to oxygenases such as P-450 cytochromes, suggests that FdVI may have a similar role in R. capsulatus.The photosynthetic bacterium Rhodobacter capsulatus is a facultative phototroph endowed with remarkable abilities of metabolic adaptation. It can grow autotrophically or heterotrophically either in the light or in darkness and can choose between five different growth modes (Madigan and Gest, 1979). It is also capable of fixing molecular nitrogen through a metabolic process which has been extensively studied by biochemical (Hallenbeck et al., 1982a; and genetic approaches (Willison et al., 1985;Klipp et al., 1988). Nitrogen fixation is catalyzed by nitrogenase and requires ATP and a low-potential reductant. Two types of electron-carrier proteins, ferredoxins (Fd) and flavodoxins, are known to serve as electron donors to nitrogenase. In R. capsulatus, the identification of the actual physiological reductant of nitrogenase is complicated by the occurence in this bacterium of an exceptional diversity of electron carriers. Five distinct ferredoxins have been isolated and biochemically characterized; three of them, FdI, FdII and FdIII, appear as representative molecular forms of the dicluster ferredoxins found in a wide range of bacteria (Bruschi and Guerlesquin, 1988). FdI and the homodimeric FdIII, both contain two [4Fe-4S] clusters/monomer (Hallenbeck et al.,

In Rhodobacter capsulatus, ferredoxin I (FdI) serves as natural electron donor to nitrogenase. In order to probe amino acid residues possibly involved in the interaction with dinitrogenase reductase, FdI was subjected to site-specific mutagenesis. A three-dimensional structure of Fdl was designed by computer modelling and used for selecting target residues. Mutant ferredoxins bearing substitutions of surface residues, as well as a variant having a Met2-Tyr replacement in the vicinity of one cluster, have been constructed. All FdI variants were expressed to similar levels both in Escherichia coli and in a FdIdeleted mutant of the natural host. Once purified, the mutant ferredoxins exhibited molecular and spectroscopic properties almost identical to wild-type FdI. Determination of the reduction potential of FdI by cyclic voltammetry gave an EA of -510 mV (pH 7.6) for both clusters, which is one of the lowest values reported for a 2[4Fe-4S] ferredoxin. Only the [Tyr2]FdI variant showed a significant difference in redox potential (AE', = -15 mV). Based on in vitro assays, a [Glu27, Glu281FdI double mutant exhibited a twofold decrease in the electron transfer rate to dinitrogenase reductase while the affinity of this mutant for the enzyme was barely affected. On the other hand, an Asp36-.His substitution resulted in a sevenfold increase of the apparent K,,, for dinitrogenase reductase. Unlike FdI and the other mutant ferredoxins, the [His36]FdI variant also failed to form a cross-linked complex with dinitrogenase reductase upon incubation with a carbodiimide. It is concluded that Asp36 in FdI probably participates in the interaction between the two protein partners. Nevertheless, all the FdI mutants proved competent in restoring a wild-type phenotype when expressed in a FdI-deleted mutant background, indicating that none of the studied residues was absolutely critical for electron transfer to nitrogenase.

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