Catalysis-dependent selenium incorporation and migration in the nitrogenase active site iron-molybdenum cofactor

Abstract

Dinitrogen reduction in the biological nitrogen cycle is catalyzed by nitrogenase, a two-component metalloenzyme. Understanding of the transformation of the inert resting state of the active site FeMo-cofactor into an activated state capable of reducing dinitrogen remains elusive. Here we report the catalysis dependent, site-selective incorporation of selenium into the FeMo-cofactor from selenocyanate as a newly identified substrate and inhibitor. The 1.60 Å resolution structure reveals selenium occupying the S2B site of FeMo-cofactor in the Azotobacter vinelandii MoFe-protein, a position that was recently identified as the CO-binding site. The Se2B-labeled enzyme retains substrate reduction activity and marks the starting point for a crystallographic pulse-chase experiment of the active site during turnover. Through a series of crystal structures obtained at resolutions of 1.32–1.66 Å, including the CO-inhibited form of Av1-Se2B, the exchangeability of all three belt-sulfur sites is demonstrated, providing direct insights into unforeseen rearrangements of the metal center during catalysis.DOI: http://dx.doi.org/10.7554/eLife.11620.001

Evaluation of the Catalytic Relevance of the CO‐Bound States of V‐Nitrogenase

“…Further, it provides the initial biochemical evidence for the potential relevance of the belt Fe atoms to N 2 reduction, which aligns well with results of the previous spectroscopic and structural analyses of CO binding. [14,29,30] Further exploration of the utility of the catalytically competent, CO-bound conformation of V-nitrogenase, along with H/D exchange experiments and a more extensive EPR study aiming to unveil the fine structure of the CO-bound state, is currently underway in hopes of gaining relevant insights into the catalytic mechanism of CO reduction by nitrogenase.…”

Nitrogenase Cofactor: Inspiration for Model Chemistry

“…Fe−C bond elongation has been indicated by extended X‐ray absorption fine structure (EXAFS) and nuclear resonance vibrational spectroscopy (NRVS) . On the other hand, the experimentally demonstrated replacement of the μ 2 ‐sulfide S2B sulfide replacement by CO or selenocyanate established that a reversible Fe−S bond cleavage is possible (Figure B), as did the observation of Fe−S dissociation in smaller Fe−S clusters . In contrast, the endo coordination (Figure C) was supported by electron nuclear double resonance (ENDOR) experiments …”

Negative Impact of Total Body Irradiation on the Antitumor Activity of Rhenium-(I)-diselenoether

“…During the course of the experiment, animals were to be sacrificed by cervical dislocation under anesthesia if any of the following signs occurred: signs of suffering (cachexia, weakening), treatment-related toxicity (hunching, convulsions), tumor growing to 10% of body weight, tumor ulcerating and remaining open, position of tumor interfering with movement/feeding,15% body weight loss for 3 consecutive days or 20% body weight loss for 1 day, clinical signs of tumor development (hindback paralysis, failure to groom, restlessness, abnormal posture or changes in resting posture, loss of mobility, paralysis etc.). Animals were sacrificed when the subcutaneous tumors reached a maximum volume of 2000 mm 3 . It was for this latter reason that treatments were stopped at day 52 and animals were sacrificed on day 53.…”

“…In that case, the presence of Se in the active site of hydrogenase enhanced its enzymatic property. Selective incorporation of Se into the active site of the molybdenum-iron (Mo-Fe) protein is essential for the catalytical effect of nitrogenase (3). Ironselenium ferredoxin with two clusters [4Fe-4Se] is present in the active site of a hydrogenase (4), with the same role as the iron-sulfur ferredoxin as a two-electron carrier.…”