Electrochemical titration of the S = 32 and S = 12 states of the iron protein of nitrogenase

AbstractThe iron protein of nitrogenase delivers electrons and ATP to the iron-molybdenum protein, which in turn reduces dinitrogen to ammonia. The iron protein contains a single four-iron, four-sulfur prosthetic group, detectable by ESR spectroscopy m its reduced form. Until recently, spin quantitations suggested that only a portion of the reduced iron protein was being detected by ESR, but recent work in several laboratories has shown that there is a spin = 32 signal near g = 5 in addition to the well characterized spin = 12 signal near g = 1.94. In this paper we characterize the redox properties of both states in the presence and absence of ATP and ADP, and find that the new spin state has identical redox properties to those previously determined for the spin = 12 state

Electron paramagnetic resonance studies on nitrogenase. III. Function of magnesium adenosine 5'-triphosphate and adenosine 5'-diphosphate in catalysis by nitrogenase

The electron paramagnetic resonance spectra of azoferredoxin and molybdoferredoxin, components of the nitrogenase of Clostridium pasteurianum, disappear when the proteins are oxidized by certain dyes. When molybdoferredoxin and azoferredoxin were mixed in a 1 to 2 molar ratio, the electron paramagnetic resonance spectrum of the mixture was the sum of the two spectra with the exception of a slight change in the azoferredoxin signal. Addition of magnesium ATP and dithionite to this reconstituted nitrogenase resulted in a rapid change in the spectrum of both nitrogenase components; the molybdoferredoxin spectrum at all g-values decreased with a half-life less than 70 ms to 40% of its original size whereas the azoferredoxin signal changed in shape and size with a half-life of less than 40 ms. If an ATP-generating system was added instead of MgATP so that no ADP accumulated, then the molybdoferredoxin signal almost completely disappeared and the azoferredoxin signal changed in shape and slightly in size. These changes occurred at molar ratios of molybdoferredoxin to azoferredoxin from 1:14 to 1:0.2. If the reaction was allowed to consume the reductant, then the molybdoferredoxin signal(s) was restored but the azoferredoxin signal disappeared. The signal of azoferredoxin was restored and the signal of molybdoferredoxin again disappeared on addition of more reductant. The data suggest that for nitrogenase to catalyze the reduction of substrates, the magnesium ATP-reduced azoferredoxin complex is formed first and this complex then reacts with molybdoferredoxin to allow electron flow. In addition the data suggests that the rate-limiting reaction is an ATP-mediated electron flow from azoferredoxin to molybdoferredoxin. Finally the results show that no flow of electrons from azoferredoxin or molybdoferredoxin occurs when a mixture of ADP and ATP in a molar ratio of 2:1 is added initially or is reached by conversion of ATP to ADP and inorganic phosphate during reduction of protons. A mechanism consistent with these findings is proposed.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/33941/1/0000208.pd

Electron paramagnetic resonance studies on nitrogenase. II. Interaction of adenosine 5'-triphosphate with azoferredoxin

The interaction of ATP with both iron-sulfur proteins of nitrogenase from Clostridium pasteurianum, azoferredoxin and molybdoferredoxin, has been studied by low-temperature EPR spectroscopy. ATP in the presence of Mg2+ changes the rhombic EPR signal of azoferredoxin with g-values of 2.06, 1.94 and 1.87 to an axial signal, with g values of 2.04 and 1.93. The binding of two molecules of ATP per azoferredoxin dimer (mol. wt 55 000) is suggested. Comparative data with other purine and pyrimidine nucleotides and ATP analogues demonstrate the involvement of structural elements of the substrate in the conversion of the EPR signal of azoferredoxin. A similar effect is induced by 5 M urea, which suggests that ATP causes a conformation change of the protein. In contrast, no effect of ATP was observed on the EPR signal of molybdoferredoxin.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/33940/1/0000207.pd

On the structure and function of nitrogenase from W5

Molybdoferredoxin from W5 was fractionated into MoFd with two atoms of molybdenum per 220,000 daltons and a specific activity of 2.6 [mu]moles C2H2 reduced/min/mg protein and into a catalytically inactive species with an identical protein moiety but an incomplete active centre. Native MoFd is a tetramer composed of two 50,000 and two 60,000 dalton subunits. At low protein concentrations the tetramer is in equilibrium with a dimer. Under low ionic strength and at low pH further dissociation into monomers occurs. MoFd and azoferredoxin have distinct electron paramagnetic resonance spectra. The EPR spectrum of AzoFd and that of the combination of the two nitrogenase components undergoes characteristic changes upon addition of MgATP2-.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34037/1/0000314.pd

Kinetic and Spectroscopic Analysis of the Inactivating Effects of Nitric Oxide on the Individual Components of Azotobacter vinelandii Nitrogenase

The effects of nitric oxide (NO) on the individual components of Azotobacter vinelandii nitrogenase have been examined by kinetic and spectroscopic methods. Incubation of the Fe protein (Av2) for 1 h with stoichiometries of 4- and 8-fold molar excesses of NO to Av2 dimer resulted in a complete loss of activity of Av2 in C2H2-reduction assays. The kinetics of inactivation indicated that the minimum stoichiometry of NO to Av2 required to fully inactivate Av2 lies between 1 and 2. The rate of inactivation of Av2 activity by NO was stimulated up to 2-fold by the presence of MgATP and MgADP but was unaffected by the presence of sodium dithionite. Unexpectedly, complete inactivation of Av2 by low ratios of NO to Av2 also resulted in a complete loss of its ability to bind MgATP and MgADP. UV-visible spectroscopy indicated that the effect of NO on Av2 involves oxidation of the [4Fe-4S] center. EPR spectroscopy revealed that the loss of activity during inactivation of Av2 by NO correlated with the loss of the S = 1/2 and S = 3/2 signals. Appearance of the classical and intense iron-nitrosyl signal (g = 2.03) was only observed when Av2 was incubated with large molar excesses of NO and the appearance of this signal did not correlate with the loss of Av2 activity. The effects of NO on the MoFe protein (Avl) were more complex than for Av2. A time-dependent inactivation of Avl activity (C2H2 reduction) was observed which required considerably higher concentrations of NO than those required to inactivate Av2 (up to 10 P a ) . In addition, the effects of NO on Avl were significantly affected by the presence of sodium dithionite. In fact, kinetic evidence suggests that an Avl-catalyzed, NO-dependent consumption of dithionite occurs before Avl is inactivated by NO. A correlation between UV-visible and EPR spectral features and the extent of NO inactivation has been established. The inactivation of either nitrogenase component by NO did not lead to aggregation or dissolution into their constitutive subunits. However, NO inactivation did cause changes in both proteins since neither NO-treated protein inhibited C2H2-reducing activity in assays containing equimolar concentrations of untreated protein. The effects of NO on both nitrogenase components are interpreted in terms of the known reactivity of NO with Fe-S centers