Comparative biochemical characterization of the iron-only nitrogenase and the molybdenum nitrogenase from Rhodobacter capsulatus

The component proteins of the iron-only nitrogenase were isolated from Rhodobacter capsulatus (Delta nifHDK, Delta modABCD strain) and purified in a one-day procedure that included only one column-chromatography step (DEAE-Sephacel). This procedure yielded component 1 (FeFe protein, Rc1(Fe)), which was more than 95% pure, and an approximately 80% pure component 2 (Fe protein, Rc2(Fe)). The highest specific activities, which were achieved at an Rc2(Fe)/Rc1(Fe) molar ratio of 40:1, were 260 (C2H4 from C2H2), 350 (NH3 formation), and 2400 (H-2 evolution) nmol product formed . min(-1). mg protein(-1). The purified FeFe protein contained 26+/-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-2-producing activity, which is much less inhibited by competitive substrates than the activity of the conventional molybdenum nitrogenase. Under optimal conditions for N-2 reduction, the activity ratios (mol N-2 reduced/mol H-2 produced) obtained were 1:1 (molybdenum nitrogenase) and 1:7.5 (iron nitrogenase). The Rc1(Fe) protein has only a very low affinity for C2H2. The K-m value determined (12.5 kPa), was about ninefold higher than the K-m for Rc1(Mo) (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. Rc1(Fe), if complemented with Rc2(Mo), showed only 10-15% of the maximally possible activity. Cross-reaction experiments with isolated polyclonal antibodies revealed that Rc1(Fe) and Rc1(Mo) are immunologically not related. The most active Rc1(Fe) samples appeared to be EPR-silent in the Na2S2O4-reduced state. However, on partial oxidation with K-3[(CN)(6)] 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 Rc1(Fe) by using turnover conditions in the presence of a substrate (N-2, C2H2, H+).