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 dinitrogenase component proteins of the conventional Mo nitrogenase (MoFe protein) and of the alternative Fe-only nitrogenase (FeFe protein) were both isolated and purified from Rhodobacter capsulatus, redox-titrated according to the same procedures and subjected to an EPR spectroscopic comparison. In the course of an oxidative titration of the MoFe protein (Rc1 Mo ) three significant S ¼ 1/2 EPR signals deriving from oxidized states of the P-cluster were detected: (1) a rhombic signal (g ¼ 2.07, 1.96 and 1.83), which showed a bell-shaped redox curve with midpoint potentials (E m ) of )195 mV (appearance) and )30 mV (disappearance), (2) an axial signal (g || ¼ 2.00, g^¼ 1.90) with almost identical redox properties and (3) a second rhombic signal (g ¼ 2.03, 2.00, 1.90) at higher redox potentials (> 100 mV). While the Ôlow-potentialÕ rhombic signal and the axial signal have been both attributed to the one-electron-oxidized P-cluster (P 1+ ) present in two conformationally different proteins, the Ôhigh-potentialÕ rhombic signal has been suggested rather to derive from the P 3+ state. Upon oxidation, the FeFe protein (Rc1 Fe ) exibited three significant S ¼ 1/2 EPR signals as well. However, the Rc1 Fe signals strongly deviated from the MoFe protein signals, suggesting that they cannot simply be assigned to different P-cluster states. (a) The most prominent feature is an unusually broad signal at g ¼ 2.27 and 2.06, which proved to be fully reversible and to correlate with catalytic activity. The cluster giving rise to this signal appears to be involved in the transfer of two electrons. The midpoint potentials determined were: )80 mV (appearance) and 70 mV (disappearance). (b) Under weakly acidic conditions (pH 6.4) a slightly altered EPR signal occurred. It was characterized by a shift of the g values to 2.22 and 2.05 and by the appearance of an additional negative absorption-shaped peak at g ¼ 1.86. (c) A very narrow rhombic EPR signal at g ¼ 2.00, 1.98 and 1.96 appeared at positive redox potentials (E m ¼ 80 mV, intensity maximum at 160 mV). Another novel S ¼ 1/2 signal at g ¼ 1.96, 1.92 and 1.77 was observed on further, enzymatic reduction of the dithionite-reduced state of Rc1 Fe with the dinitrogenase reductase component (Rc2 Fe ) of the same enzyme system (turnover conditions in the presence of N 2 and ATP). When the Rc1 Mo protein was treated analogously, neither this Ôturnover signalÕ nor any other S ¼ 1/2 signal were detectable. All Rc1 Fe -specific EPR signals detected are discussed and tentatively assigned with special consideration of the reference spectra obtained from Rc1 Mo preparations.

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