title: ENDOR/HYSCORE studies of the common intermediate trapped during nitrogenase reduction of N2H2, CH3N2H, and N2H4 support an alternating reaction pathway for N2 reduction.
abstract: Enzymatic N(2) reduction proceeds along a reaction pathway composed of a sequence of intermediate states generated as a dinitrogen bound to the active-site iron-molybdenum cofactor (FeMo-co) of the nitrogenase MoFe protein undergoes six steps of hydrogenation (e(-)/H(+) delivery). There are two competing proposals for the reaction pathway, and they invoke different intermediates. In the 'Distal' (D) pathway, a single N of N(2) is hydrogenated in three steps until the first NH(3) is liberated, and then the remaining nitrido-N is hydrogenated three more times to yield the second NH(3). In the 'Alternating' (A) pathway, the two N's instead are hydrogenated alternately, with a hydrazine-bound intermediate formed after four steps of hydrogenation and the first NH(3) liberated only during the fifth step. A recent combination of X/Q-band EPR and (15)N, (1,2)H ENDOR measurements suggested that states trapped during turnover of the α-70(Ala)/α-195(Gln) MoFe protein with diazene or hydrazine as substrate correspond to a common intermediate (here denoted I) in which FeMo-co binds a substrate-derived [N(x)H(y)] moiety, and measurements reported here show that turnover with methyldiazene generates the same intermediate. In the present report we describe X/Q-band EPR and (14/15)N, (1,2)H ENDOR/HYSCORE/ESEEM measurements that characterize the N-atom(s) and proton(s) associated with this moiety. The experiments establish that turnover with N(2)H(2), CH(3)N(2)H, and N(2)H(4) in fact generates a common intermediate, I, and show that the N-N bond of substrate has been cleaved in I. Analysis of this finding leads us to conclude that nitrogenase reduces N(2)H(2), CH(3)N(2)H, and N(2)H(4) via a common A reaction pathway, and that the same is true for N(2) itself, with Fe ion(s) providing the site of reaction.
authors: Department of Chemistry, Northwestern University, Evanston, Illinois 60208, USA.
authors: Dmitriy
authors: D
authors: Lukoyanov
authors: Sergei A
authors: SA
authors: Dikanov
authors: Zhi-Yong
authors: ZY
authors: Yang
authors: Brett M
authors: BM
authors: Barney
authors: Rimma I
authors: RI
authors: Samoilova
authors: Kuppala V
authors: KV
authors: Narasimhulu
authors: Dennis R
authors: DR
authors: Dean
authors: Lance C
authors: LC
authors: Seefeldt
authors: Brian M
authors: BM
authors: Hoffman
mesh_terms: D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D006834:Hydrazines / Q000737:chemistry / Q000378:metabolism*; D007094:Imides / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D009584:Nitrogen / Q000737:chemistry / Q000378:metabolism*; D009591:Nitrogenase / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction
chemical_list: D006834:Hydrazines; D007094:Imides; D009591:Nitrogenase; C038867:diazene; D009584:Nitrogen