Electron Paramagnetic Resonance of Nitrogenase and Nitrogenase Components from Clostridium pasteurianum W5 and Azotobacter vinelandii OP

Abstract

The electron paramagnetic resonance of nitrogenase components, separately and together with the other reactants in the nitrogenase system (namely, reductant and Mg·ATP), have been examined at low temperatures (<20°K). The MoFe protein, component I or molybdoferredoxin, in the oxidized (but not oxygen-inactivated) state yields signals with g-values of 4.3, 3.7, and 2.01, and when reduced has no observable electron paramagnetic resonance. The Fe protein, component II, or azoferredoxin, yields a signal with g-values of 2.05, 1.94, and 1.89 in the reduced state that is converted by Mg·ATP into an axial signal with g-values near 2.05 and 1.94, and a second split signal near g = 4.3. The Fe protein has no definite electron paramagnetic resonance in the oxidized (not oxygen-denatured) state under these conditions. The Mg·ATP complex of reduced Fe protein reduces the MoFe protein, whereas dithionite alone does not reduce the MoFe protein. Reoxidation of the system by substrate leads to disappearance of the Fe protein signal and the reappearance of the MoFe protein signal. Thus Mg·ATP, which is hydrolyzed during substrate reduction, converts the Fe protein to a reductant capable of transferring electrons to MoFe protein, after which substrate reduction occurs.

The Fe Protein: An Unsung Hero of Nitrogenase

“…In addition, the oxidation state of the cluster also affects the extent of Fe chelation relative to that of the dithionite-reduced form of NifH [57]. As stated above, a change in the protein is also supported by the different EPR spectra of NifH observed in the presence and absence of MgATP, consistent with an apparent connection between the cluster properties and nucleotide binding [58][59][60]. The nucleotide binding behavior becomes more complicated when the MgATP hydrolysis product and nitrogenase inhibitor MgADP is taken into account.…”

Docking of nitrogenase iron‐and molybdenum‐iron proteins for electron transfer and MgATP hydrolysis: The role of arginine 140 and lysine 143 of the Azotobacter vinelandii iron protein

“…This state would be analogous to the heterologous Clostridium pasteurianurn FeP-A. vinelandii MoFeP complex that catalyzes MgATP hydrolysis, but not electron transfer (Smith et al, 1976;Emerich & Burris, 1978 (Orme-Johnson et al, 1972;Zumft et al, 1972), a change in the midpoint potential of the cluster by -150 mV (Watt et al, 1986), changes in the CD spectrum (Stephens et al, 1979), shifts in the NMR resonances of protons magnetically coupled to the paramagnetic [4Fe-4S] cluster (Meyer et al, 1988), changes in scattering of X-rays (Chen et al, 1994), and in Fez+ chelation (Walker & Mortenson, 1974). This conformation of the FeP would be required for the formation of the second, MgATP hydrolytic state, with the MoFeP.…”

On the iron-sulfur cluster in hydrogenase from Clostridium pasteurianum W5.

“…The general procedure, including the use of a vacuum manifold system for anaerobic manipulations, was as previously described (8). Samples were frozen within 1-2 sec by immersion in a rapidly stirred isopentane bath maintained at 130 K. EPR spectra were obtained at 9.2 GHz with a Varian E-9 machine fitted for low temperature operation (9).…”

“…Quantitation of spins was done by double integration of EPR signals obtained at 13 K at a nonsaturating microwave power. Comparison of hydrogenase signals to those obtained with a standard solution of copper-EDTA complex and correction for the difference in g-values yielded spin concentrations in the hydrogenase samples (8).…”