The Fe‐only nitrogenase and the Mo nitrogenase from Rhodobacter capsulatus

Abstract

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.

Die katalytische Reduktion von Distickstoff zu Ammoniak mit Molybdän: Theorie und Experiment

“…[66] Umfassende Berechnungen stützen die Vermutungen zum Mechanismus der katalytischen Reduktion von Distick- [36] Es wird daher angenommen, [51] dass das 2,6-Lutidiniumion die MoN 2 H y -Spezies über den "oberen" HIPTSubstituenten angreift und so ein Proton auf ein b-Stickstoffatom oder sogar in einer Verbindung MoNH y auf ein aStickstoffatom überträgt (die HIPT-Reste müssten sich dazu wegbiegen und verdrehen). Die Pyridinium-Säure hat einen großen Einfluss auf den Verlauf der N 2 -Reduktion: [ (11). [48,68] 3…”

“…[5] Andere Nitrogenasen enthalten Vanadium statt Molybdän (sie sind aktiv, wenn Vanadium verfügbar ist, aber ein Mangel an Molybdän herrscht) [6][7][8][9] oder Eisen (wenn weder Mo noch V verfügbar sind). [10][11][12][13] In die Erforschung der Nitrogenasen wurden bereits mehrere hundert Mannjahre Zeit investiert, und die Untersuchungen gehen weiter. Die Effizienz, mit der Ammoniak -und nicht Wasserstoff -gebildet wird, nimmt in der Reihe Mo!V!Fe drastisch ab.…”

Nitrogenase and homologs

“…While progress has been made in improving the preparations of the FeFe protein from R. capsulatus , the successful purification of an intact form of this protein has remained a challenge and the most competent form of this protein reported so far has an α 2 β 2 δ 2 -hexameric subunit composition [2]. EPR, Mössbauer and EXAFS studies of the R. capsulatus FeFe protein have provided spectroscopic evidence that this protein contains metal centers homologous to the P/P*-cluster (designated the P′-cluster) and the M/V-cluster (designated the FeFe cofactor or the Fe-cluster), respectively [58, 59]. Given the overall homology between the Fe-only nitrogenase and its Mo and V counterpart, this nitrogenase likely utilizes the same mode of action during catalysis, which involves complex formation between the two component proteins and electron transfer from the [Fe 4 S 4 ] cluster of the Fe protein, via the P′-cluster, to the Fe-cluster of the FeFe protein, where substrate reduction takes place.…”

Synthetic Analogues of the Active Sites of Nitrogenase and [NiFe] Hydrogenase

“…39 The structural analogy between the [Fe 8 S 7 ] core of 810 and the FeMo-cofactor raises a possibility that clusters 810 may be similar to the FeFe-cofactor of Fe-only nitrogenase, because the FeFe-cofactor has been suggested to be structurally similar to the FeMo-cofactor. 40 In the EPR spectrum of Feonly nitrogenase (FeFe protein) from Rhodobacter capsulatus, some states exhibit rhombic S = 1/2 signals at g = 1.96, 1.92, and 1.77 (turnover state) 41 and at g = 2.22, 2.05, 1.86 (pH 6.4, ¹5 mV vs. NHE, potential adjusted by the addition of hexacyanoferrate(III) as oxidant and Na 2 S 2 O 4 as reductant) possibly from the FeFe-cofactor, 42 and these signals happen to be similar to the EPR signals of 8 and 10.…”