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

Abstract

The component proteins of the iron-only nitrogenase were isolated from Rhodobacter capsulatus (ΔnifHDK, 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 C2H. 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+).