A Zn-immobilized metal-affinity chromatography technique was used to purify a poly-histidine-tagged, FeMo-cofactorless MoFe protein (apo-MoFe protein) from a nifB-deletion mutant of Azotobacter vinelandii. Apo-MoFe protein prepared in this way was obtained in sufficient concentrations for detailed catalytic, kinetic, and spectroscopic analyses. Metal analysis and electron paramagnetic resonance spectroscopy (EPR) were used to show that the apo-MoFe protein does not contain FeMo-cofactor. The EPR of the as-isolated apo-MoFe protein is featureless except for a minor S = 1/2 signal probably arising from the presence of either a damaged P cluster or a P cluster precursor. The apo-MoFe protein has an alpha2beta2 subunit composition and can be activated to 80% of the theoretical MoFe protein value by the addition of isolated FeMo-cofactor. Oxidation of the as-isolated apo-MoFe protein by indigodisulfonate was used to elicit the parallel mode EPR signal indicative of the two-electron oxidized form of the P cluster (P2+). The midpoint potential of the PN/P2+ redox couple for the apo-MoFe protein was shown to be shifted by -63 mV when compared to the same redox couple for the intact MoFe protein. Although the apo-MoFe protein is not able to catalyze the reduction of substrates under turnover conditions, it does support the hydrolysis of MgATP at 60% of the rate supported by the MoFe protein when incubated in the presence of Fe protein. The ability of the apo-MoFe protein to specifically interact with the Fe protein was also shown by stopped-flow techniques and by formation of an apo-MoFe protein-Fe protein complex. Finally, the two-electron oxidized form of the apo-MoFe protein could be reduced to the one-electron oxidized form (P1+) in a reaction that required Fe protein and MgATP. These results are interpreted to indicate that the apo-MoFe protein produced in a nifB-deficient genetic background [corrected] contains intact P clusters and P cluster polypeptide environments. Small changes in the electronic properties of P clusters contained within the apo-MoFe protein are most likely caused by slight perturbations in their polypeptide environments.

Improved 1H ENDOR data from the S(EPR1) intermediate formed during turnover of the nitrogenase alpha-195Gln MoFe protein with C2(1,2)H2 in (1,2)H2O buffers, taken in context with the recent study of the intermediate formed from propargyl alcohol, indicate that S(EPR1) is a product complex, likely with C2H4 bound as a ferracycle to a single Fe of the FeMo-cofactor active site. 35 GHz CW and Mims pulsed 57Fe ENDOR of 57Fe-enriched S(EPR1) cofactor indicates that it exhibits the same valencies as those of the CO-bound cofactor of the lo-CO intermediate formed during turnover with CO, [Mo4+, Fe3+, Fe6(2+), S9(2-)(d43)](+1), reduced by m = 2 electrons relative to the resting-state cofactor. Consideration of 57Fe hyperfine coupling in S(EPR1) and lo-CO leads to a picture in which CO bridges two Fe of lo-CO, while the C2H4 of S(EPR1) binds to one of these. To correlate these and other intermediates with Lowe-Thorneley (LT) kinetic schemes for substrate reduction, we introduce the concept of an "electron inventory". It partitions the number of electrons a MoFe protein intermediate has accepted from the Fe protein (n) into the number transmitted to the substrate (s), the number that remain on the intermediate cofactor (m), and the additional number delivered to the cofactor from the P clusters (p): n = m + s - p (with p = 0 here). The cofactors of lo-CO and S(EPR1) both are reduced by m = 2 electrons, but the intermediates are not at the same LT reduction stage (E(n)): (n = 2; m = 2, s = 0) for lo-CO; (n = 4; s = 2, m = 2) for S(EPR1). This is the first proposed correlation of an LT E(n) kinetic state with a well-defined chemical state of the enzyme.