Comparative biochemical characterization of the iron-only nitrogenase and the molybdenum nitrogenase from <em>Rhodobacter capsulatus</em>.

The component proteins of the iron-only nitrogenase were isolated from Rhodobacter capsulatus (ΔniƒHDK, ΔmodABCD 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 (H₂ 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 H₂-producing activity, which is much less inhibited by competitive substrates than the activity of the conventional molybdenum nitrogenase. Under optimal conditions for N₂ reduction, the activity ratios (mol N₂ reduced/mol H₂ 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 K₃[Fe(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 (N₂, C₂H₂, H⁺). [ABSTRACT FROM AUTHOR]