Analysis of early intermediate states of the nitrogenase reaction by Se incorporation and regularization of EPR spectra

Abstract

Abstract Due to the complexity of the catalytic FeMo cofactor site in nitrogenases that mediates the reduction of molecular nitrogen to ammonium, mechanistic details of this reaction remain under debate. In this study, selenium- and sulfur-incorporated FeMo cofactors of the catalytic MoFe protein component from Azotobacter vinelandii were prepared under turnover conditions and investigated by using different EPR methods. Complex signal patterns were observed in the continuous wave EPR spectra of selenium-incorporated samples, which were analyzed by Tikhonov regularization, a method that has not yet been applied to high spin systems of transition metal cofactors, and by an already established grid-of-error approach. Both methods yielded similar probability distributions that revealed the presence of at least four other species with different electronic structures in addition to the ground state E0. Some of these species were preliminary assigned to hydrogenated E2 states. In addition, advanced pulsed-EPR experiments were utilized to verify the incorporation of sulfur and selenium into the FeMo cofactor, and to assign hyperfine couplings of 33S and 77Se that directly couple to the FeMo cluster. With this analysis, we report selenium incorporation under turnover conditions as a straightforward approach to stabilize and analyze early intermediate states of the FeMo cofactor.