Electron Inventory, Kinetic Assignment (En), Structure, and Bonding of Nitrogenase Turnover Intermediates with C2H2 and CO

Abstract

Improved ¹H ENDOR data from the S_EPR1 intermediate formed during turnover of the nitrogenase α-195^Gln MoFe protein with C₂^1,2H₂ in ^1,2H₂O 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⁺, Fe₆²⁺, 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.