The component proteins of the iron-only nitrogenase were isolated from Rhodobacter capsulatus (AnifhTDK, AmodABCD strain) and purified in a one-day procedure that included only one columnchromatography step (DEAE-Sephacel). This procedure yielded component 1 (FeFe protein, RclFe), which was more than 95% pure, and an approximately 80% pure component 2 (Fe protein, R c~~' ) . The highest specific activities, which were achieved at an R c~~V R C~~~ molar ratio of 40: 1, were 260 (C,H, from C,H,), 350 (NH, formation), and 2400 (H, evolution) nmol product formed . min-' . mg protein-'.The purified FeFe protein contained 26 -t 4 Fe atoms ; it did not contain Mo, V, or any other heterometal atom.The most significant catalytic property of the iron-only nitrogenase is its high H,-producing activity, which is much less inhibited by competitive substrates than the activity of the conventional molybdenum nitrogenase. Under optimal conditions for N, reduction, the activity ratios (mol N, reduced/mol H, produced) obtained were 1 : 1 (molybdenum nitrogenase) and 1 :7.5 (iron nitrogenase). The Rcl" protein has only a very low affinity for C,H,. The K,, value determined (12.5 kPa), was about ninefold higher than the K,, for RclM" (1.4 kPa). The proportion of ethane produced from acetylene (catalyzed by the iron nitrogenase), was strictly pH dependent. It corresponded to 5.5% of the amount of ethylene at pH 6.5 and was almost zero at pH values greater than 8.5.In complementation experiments, component 1 proteins coupled very poorly with the 'wrong' component 2. RclF', if complemented with R c~~" , showed only 10-15% of the maximally possible activity. Cross-reaction experiments with isolated polyclonal antibodies revealed that Rcl'" and Rc lM" are immunologically not related.The most active RclFe samples appeared to be EPR-silent in the Na,S,O,-reduced state. However, on partial oxidation with K,[Fe(CN),] or thionine several signals occurred. The most significant signal appears to be the one at g = 2.27 and 2.06 which deviates from all signals so far described for P clusters. It is a transient signal that appears and disappears reversibly in a redox potential region between -100 mV and + 150 mV. Another novel EPR signal (g = 1.96, 1.92, 1.77) occurred on further reduction of RclF" by using turnover conditions in the presence of a substrate (N,, C,H,, H+).Keywords: nitrogenase ; iron protein ; cofactor; EPR; Rhodobacter capsulatus.Three genetically distinct types of nitrogenase systems (ng vnJ unf, have so far been proved to exist in nature. The most widespread and intensively characterized system is the classical Mo-containing nitrogenase (nif system) found in all diazotrophs [l, 21. During the last few years, two types of alternative, Moindependent nitrogenases have been discovered and described. One is a vanadium-containing nitrogenase (vnf system) (for review see [3]) and the other enzyme lacks both Mo and V (anf system) and has been tentatively designated as Fe nitrogenase [4][5][6]. Although there is much circumstantial and...

The photosynthetic bacterium Rhodobacter capsulatus has, in addition to the Mo nitrogenase, a second Mo-independent nitrogen-fixing system, an 'iron-only' nitrogenase which is strongly repressed by molybdate. The Mo0,2-concentration causing 50 % repression of the alternative nitrogenase in nijHDK-cells was 6 nM. If MOO:-was added to a growing nijHDK-culture which had already expressed the alternative nitrogenase, the production of ethane from acetylene, by whole cells, was stimulated dramatically. In spite of the fact that C,H, formation decreased continuously during the duration of the experiment (3 days), the total C,H, production increased about twofold within the first 24 h, whereas the relative yield of C,H, increased from 2 % (C,H$C,H, X 100) in the absence of MOO:-, to a maximal value of 69% in the presence of MOO:-(1 mM) after 72 h incubation. This 'Mo effect' appeared to be stronger the higher the MOO:-concentration in the medium and the longer the incubation time. In the presence of ReO;, WO2-or VO:-, a similar effect did not occur.The 'Mo effect' was not observed in a nijHDK-nijZ-double mutant which is unable to synthesize the FeMo cofactor and was diminished in a nijHDK-nifQ-mutant.Crude extracts from nifHDK-cells cultivated in the presence of MOO:-, also showed enhanced production of ethane. Component 1, purified from those extracts, displayed an S = 3/2 EPR signal which was relatively weak but characteristic for the FeMoco. These results strongly support the suggestion that the 'Mo effect' is a consequence of the formation of a hybrid enzyme consisting of the apoprotein of the alternative nitrogenase and the FeMo cofactor of the conventional nitrogenase.The 'Mo effect' was not influenced by the addition of chloramphenicol to the cultures. The occurrence of the 'Mo effect' appeared, therefore, to be independent of de-novo protein synthesis. The analysis of niP-lacZ and nijiV-lacZ fusions proved that both genes necessary for the FeMo cofactor synthesis are also expressed under conditions of MOO:-deficiency.The possible explanations for incorporation of the FeMoco into component 1 of the alternative nitrogenase are discussed.As the metal clusters of nitrogenases ('P' cluster, FeMo cofactor) play a key role in the process of the biological nitrogen fixation, information about their properties and biosynthesis seems to be of significance (Muller and Newton, 1983;Burgess, 1990). The type of cofactor (FeMoco, FeVco, FeFeco) and also the protein environment of this cluster influence the activity and the substrate selectivity of the nitrogenase (Scott et al., 1990;Miiller et al., 1992).The conventional FeMoco-containing nitrogenases have been characterized to reduce acetylene exclusively to ethylene. In a recent work, Ashby and colleagues (1987) demonstrated that the Mo nitrogenase of Klebsiella pneumoniae is

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