Diazene (HNNH) Is a Substrate for Nitrogenase:  Insights into the Pathway of N₂ Reduction

Abstract

Nitrogenase catalyzes the sequential addition of six electrons and six protons to a N 2 that is bound to the active site metal cluster FeMo-cofactor, yielding two ammonia molecules. The nature of the intermediates bound to FeMo-cofactor along this reduction pathway remains unknown, although it has been suggested that there are intermediates at the level of reduction of diazene (HN=NH, also called diimide) and hydrazine (H 2 N-NH 2 ). Through in situ generation of diazene during nitrogenase turnover, we show that diazene is a substrate for the wild-type nitrogenase and is reduced to NH 3 . Diazene reduction, like N 2 reduction, is inhibited by H 2 . This contrasts with the lack of H 2 inhibition when nitrogenase reduces hydrazine. These results support the existence of an intermediate early in the N 2 reduction pathway at the level of reduction of diazene. Freeze-quenching a MoFe protein variant with α-195 His substituted by Gln and α-70 Val substituted by Ala during steady-state turnover with diazene resulted in conversion of the S = 3/2 resting state FeMo-cofactor to a novel S = 1/2 state with g 1 = 2.09, g 2 = 2.01, and g 3 ~ 1.98. 15 N-and 1 H-ENDOR establish that this state consists of a diazene-derived [-NH x ] moiety bound to FeMo-cofactor. This moiety is indistinguishable from the hydrazine-derived [-NH x ] moiety bound to FeMo-cofactor when the same MoFe protein is trapped during turnover with hydrazine. These observations suggest that diazene joins the normal N 2 -reduction pathway, and that the diazene-and hydrazine-trapped turnover states represent the same intermediate in the normal reduction of N 2 by nitrogenase. The implications of these findings for the mechanism of N 2 reduction by nitrogenase are discussed. KeywordsFeMo-cofactor; EPR; ENDOR; Active Site; Substrate Nitrogenase is the enzyme responsible for catalyzing biological reduction of N 2 to two NH 3 , an essential reaction in the global biogeochemical nitrogen cycle (1-3). The minimum stoichiometry for the nitrogenase catalyzed reduction of N 2 involves delivery of 8e − and 8H + (eqn 1). † This work was supported by grants from the National Institutes of Health (R01-GM59087 to LCS and DRD; HL13531 to BMH), the National Science Foundation (MCB-0316038 to BMH) and the United States Department of Agriculture Postdoctoral Fellowship program (2004-35318-14905 to BMB).*Address correspondence to these authors: LCS, phone (435) 797-3964, fax (435) email seefeldt@cc.usu.edu; DRD, phone (540) 231-5895, fax (540) email deandr@vt.edu; BMH, phone (847) (Figure 1).Relatively little is known at a molecular level about the nitrogenase N 2 -reduction mechanism beyond the fact that N 2 binds to and is reduced at one or more of the metal atoms of FeMocofactor ( Figure 1) Both the Chatt and Schrock cycles belong to one fundamental class of potential nitrogenase mechanismsin which the first three 'H-atoms' (e − /H + ) are sequentially added to a single N atom, in those instances the distal N of an end-on bound N 2 , followed by cleava...

Unification of reaction pathway and kinetic scheme for N 2 reduction catalyzed by nitrogenase

“…Key to this issue is the finding that H 2 inhibits the reduction of diazene (22), but not hydrazine (23). We take that to mean that H 2 and diazene bind competitively, as do H 2 and N 2 (2,5,6), but that this is not the case for hydrazine.…”

“…Sample preparation followed freeze-quench procedures previously described (22,24,25); the 35 GHz CW (26) and pulsed (27) EPR spectrometers also have been described. Fourier transforms were performed with Bruker software, Win-EPR.…”

Carbon dioxide reduction to methane and coupling with acetylene to form propylene catalyzed by remodeled nitrogenase

“…All other gases were purchased from Air Liquide. ) were grown, and the corresponding nitrogenase MoFe proteins were expressed and purified as described (49). All MoFe proteins in this study contain a seven-His tag addition near the carboxyl-terminal end of the α-subunit.…”

Late Metal Scaffolds that Activate Both, Dinitrogen and Reduced Dinitrogen Species NxHy

“…The fact that diazene and hydrazine have been shown to represent substrates of nitrogenase provides further support for this mechanism. [6,9,[30][31][32] Furthermore both of these intermediates are released upon acid or base hydrolysis of the enzyme under turnover conditions. [6,31,33,34] Moreover, the recent discovery of iron containing model complexes that cleave N 2 [35,36] or even fix N 2 catalytically [37] strengthen the argument for N 2 activation and conversation at the iron site in the FeMo-co.…”