Open AccessThe Fe‐only nitrogenase and the Mo nitrogenase from Rhodobacter capsulatus
Author(s) -
Siemann Stefan,
Schneider Klaus,
Dröttboom Melanie,
Müller Achim
Publication year - 2002
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.2002.02804.x
Subject(s) - nitrogenase , rhodobacter , redox , chemistry , electron paramagnetic resonance , redox titration , cluster (spacecraft) , crystallography , stereochemistry , inorganic chemistry , biochemistry , nuclear magnetic resonance , organic chemistry , physics , nitrogen fixation , mutant , computer science , nitrogen , gene , programming language
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 = 1/2 EPR signals deriving from oxidized states of the P‐cluster were detected: (1) a rhombic signal ( g = 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 || = 2.00, g ⊥ = 1.90) with almost identical redox properties and (3) a second rhombic signal ( g = 2.03, 2.00, 1.90) at higher redox potentials (> 100 mV). While the ‘low‐potential’ 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 ‘high‐potential’ rhombic signal has been suggested rather to derive from the P 3+ state. Upon oxidation, the FeFe protein (Rc1 Fe ) exibited three significant S = 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 = 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 = 1.86. (c) A very narrow rhombic EPR signal at g = 2.00, 1.98 and 1.96 appeared at positive redox potentials ( E m = 80 mV, intensity maximum at 160 mV). Another novel S = 1/2 signal at g = 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 ‘turnover signal’ nor any other S = 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.