TY - JOUR
T1 - ENDOR/HYSCORE studies of the common intermediate trapped during nitrogenase reduction of N 2H 2, CH 3N 2H, and N 2H 4 support an alternating reaction pathway for N 2 reduction
AU - Lukoyanov, Dmitriy
AU - Dikanov, Sergei A.
AU - Yang, Zhi Yong
AU - Barney, Brett M.
AU - Samoilova, Rimma I.
AU - Narasimhulu, Kuppala V.
AU - Dean, Dennis R.
AU - Seefeldt, Lance C.
AU - Hoffman, Brian M.
PY - 2011/8/3
Y1 - 2011/8/3
N2 - 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 15N, 1,2H 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 xH 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 14/15N, 1,2H ENDOR/HYSCORE/ESEEM measurements that characterize the N-atom(s) and proton(s) associated with this moiety. The experiments establish that turnover with N 2H 2, CH 3N 2H, and N 2H 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 2H 2, CH 3N 2H, and N 2H 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.
AB - 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 15N, 1,2H 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 xH 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 14/15N, 1,2H ENDOR/HYSCORE/ESEEM measurements that characterize the N-atom(s) and proton(s) associated with this moiety. The experiments establish that turnover with N 2H 2, CH 3N 2H, and N 2H 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 2H 2, CH 3N 2H, and N 2H 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.
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U2 - 10.1021/ja2036018
DO - 10.1021/ja2036018
M3 - Article
C2 - 21744838
AN - SCOPUS:79960851352
SN - 0002-7863
VL - 133
SP - 11655
EP - 11664
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
IS - 30
ER -