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Mechanism of nitrogen fixation by nitrogenase: the next stage.

Hoffman BM, Lukoyanov D, Yang ZY, Dean DR, Seefeldt LC - Chem. Rev. (2014)

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, Utah State University , 0300 Old Main Hill, Logan, Utah 84322, United States.

ABSTRACT

Two major pointscan be made regarding intermediates trapped: (1)Characterization of the E4 “Janus” intermediateas bearing four reducing equivalents in the form of two [Fe–H–Fe]bridging hydrides has provided the foundation for proposals that theFeMo-co core is never oxidized or reduced by more than one equivalentrelative to the resting-state, and that the oxidative couple in factis operative, Figure 19, I. (2) The characterizationof the common intermediates H and I, trappedduring turnover with nitrogenous substrates, led to the proposed unificationof kinetic scheme and A reaction pathway, Figure 12.

Reductive eliminationof two hydrides upon N2 binding (re mechanism) providesan explanation for the nitrogenase stoichiometry (eq 1) and for the obligatory formation of H2 upon N2 binding. This mechanism for H2 production uponN2 binding to E4, Figure 13, lower, satisfies both the stoichiometric constraint of HD formation(Chart 1, line i) and the “T+” constraint against exchange of gas-derived hydrons withsolvent (Chart 1, line ii), whereas the hpmechanism (Figure 13, upper) satisfies neither.The re mechanism further involves D2 binding to a stateat the “diazene level” of reduction, as required bythe constraint of eq 3 and Chart 1, line iii. Finally, to the best of our knowledge, all otherconstraints on the mechanism, most of which are not directly tiedto D2 binding, are satisfied, as well.

This mechanismanswers the following long-standing and oft-repeated question: Whydoes nature “waste” four ATP/two reducing equivalentsthrough an obligatory loss of H2 when N2 binds?The answer follows: reductive elimination of H2 upon bindingof N2 to FeMo-co of the E4 state generates astate in which highly reduced FeMo-co binds N2, which likelyis activated for reduction through electrostatic interactions withthe remaining two sulfur-bound protons. Transfer of the two reducingequivalents generated by the reductive elimination, combined withtransfer of the two activating protons, then forms N2H2, Figure 13, lower, in keeping withthe P–A scheme of Figure 12. It appearsthat only through this activation is the enzyme able to hydrogenateN2.

This mechanism has been supported by a rigorous test which providedexperiments in which C2H2 is added to an N2/D2 reaction mixture. Although diatomic D2 does not reduce nitrogenase C2H2 in the absenceof N2, the re mechanism successfully predicted that turnoverunder C2H2/D2/N2 wouldbreach the separation of gaseous D2 from solvent protonsby generating both C2H3D andC2H2D2.

The conclusions regardingH2 formation upon N2 binding reached from thisstudy are as follows. (i) The unprecedented incorporation of D fromD2 into the nitrogenase reduction products C2H2D2 and C2H3D duringturnover under C2H2/D2/N2 in H2O demonstrates the presence of the E4(2D) and E2(D) states under these conditions. In our viewany model that fails to incorporate obligatory H2 lossas a fundamental aspect of N2 activation is unlikely toprovide a robust description of the chemistry associated with thebiological process.242 (ii) This incorporationprovides a very clear demonstration of the essential mechanistic rolefor obligatory, reversible loss of H2 upon N2 binding and thus of the eight-electron stoichiometry for nitrogenfixation by nitrogenase embodied in eq 1. Untilnow, the data indicating that some H2 must be evolved duringN2 reduction has been viewed as being much more compellingthan the data indicating an obligatory evolution of one H2 for every N2 reduced, leading to the stoichiometry ofeq 1.17 (iii) Theformation of E4(2D) and E2(D) during turnoverunder D2/N2 in H2O is predicted bythe re mechanism for the activation of FeMo-cofactor for reductionof N2, and the interception of these intermediates by C2H2 thus provides direct experimental evidence insupport of this mechanism (Figure 17). (iv)The well-known reduction of protons by D2 to form 2HD duringturnover under D2/N2 in H2O and thenewly discovered reductions of C2H2 by D2/N2 should be viewed as being catalyzed by nitrogenasewith N2 as cocatalyst. (v) This review has proposed anexplanation of the inability of H2/D2 to reducenitrogenase and/or catalyze substrate reduction in the absence ofN2.

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Formationof deuterated acetylenes during turnover under N2/D2/C2H2 as predicted accordingto re mechanism. Cartoons again depict the Fe2,3,6,7 face of resting-stateFeMo-co, with no attempt to incorporate likely structural modifications.Figure shows that the “reverse” of re mechanism throughdisplacement of N2 by D2 produces, successively,E4(2D) and E2(D), further showing potentialreaction channels for capture of E4(2D) and E2(D) intermediates with C2H2.
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fig15: Formationof deuterated acetylenes during turnover under N2/D2/C2H2 as predicted accordingto re mechanism. Cartoons again depict the Fe2,3,6,7 face of resting-stateFeMo-co, with no attempt to incorporate likely structural modifications.Figure shows that the “reverse” of re mechanism throughdisplacement of N2 by D2 produces, successively,E4(2D) and E2(D), further showing potentialreaction channels for capture of E4(2D) and E2(D) intermediates with C2H2.

Mentions: With this foundation,we recognized that the re mechanism predicts that turnover under C2H2/D2/N2 should not onlyincorporate H from solvent to generate C2H4 bythe normal reduction process, but through the agency of the addedN2 also should breach the separation of gaseous D2 from solvent protons by generating both C2H3D and C2H2D2 (Figure 15). According to the re mechanism, when turnoveris carried out under N2/D2, D2 canreact with E4(N2/N2H2),replacing the N2 and undergoing oxidative addition to generateE4(2D). We recognized that this state in fact might beexpected to react with C2H2 to form C2H2D2 through the idealized mechanism (Figure 16A) involving terminalization of an [Fe–D–Fe]bridge of E4, and migratory insertion of bound C2H2 into the Fe–D bond to form an Fe-alkenyl intermediate,followed by reductive elimination of C2H2D2.195,231 Previous studies17,103 could not distinguish reaction at the E4 state from reactionat the E2 state when C2H2 is reducedin the absence of N2, as N2 is required to enablegaseous D2 to enter the nitrogenase catalytic process.The possibility that acetylene can access different nitrogenase redoxstates, however, had been suggested on the basis of experiments usinga nitrogenase variant that exhibits N2 reduction that isresistant to inhibition by acetylene.232,233


Mechanism of nitrogen fixation by nitrogenase: the next stage.

Hoffman BM, Lukoyanov D, Yang ZY, Dean DR, Seefeldt LC - Chem. Rev. (2014)

Formationof deuterated acetylenes during turnover under N2/D2/C2H2 as predicted accordingto re mechanism. Cartoons again depict the Fe2,3,6,7 face of resting-stateFeMo-co, with no attempt to incorporate likely structural modifications.Figure shows that the “reverse” of re mechanism throughdisplacement of N2 by D2 produces, successively,E4(2D) and E2(D), further showing potentialreaction channels for capture of E4(2D) and E2(D) intermediates with C2H2.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4012840&req=5

fig15: Formationof deuterated acetylenes during turnover under N2/D2/C2H2 as predicted accordingto re mechanism. Cartoons again depict the Fe2,3,6,7 face of resting-stateFeMo-co, with no attempt to incorporate likely structural modifications.Figure shows that the “reverse” of re mechanism throughdisplacement of N2 by D2 produces, successively,E4(2D) and E2(D), further showing potentialreaction channels for capture of E4(2D) and E2(D) intermediates with C2H2.
Mentions: With this foundation,we recognized that the re mechanism predicts that turnover under C2H2/D2/N2 should not onlyincorporate H from solvent to generate C2H4 bythe normal reduction process, but through the agency of the addedN2 also should breach the separation of gaseous D2 from solvent protons by generating both C2H3D and C2H2D2 (Figure 15). According to the re mechanism, when turnoveris carried out under N2/D2, D2 canreact with E4(N2/N2H2),replacing the N2 and undergoing oxidative addition to generateE4(2D). We recognized that this state in fact might beexpected to react with C2H2 to form C2H2D2 through the idealized mechanism (Figure 16A) involving terminalization of an [Fe–D–Fe]bridge of E4, and migratory insertion of bound C2H2 into the Fe–D bond to form an Fe-alkenyl intermediate,followed by reductive elimination of C2H2D2.195,231 Previous studies17,103 could not distinguish reaction at the E4 state from reactionat the E2 state when C2H2 is reducedin the absence of N2, as N2 is required to enablegaseous D2 to enter the nitrogenase catalytic process.The possibility that acetylene can access different nitrogenase redoxstates, however, had been suggested on the basis of experiments usinga nitrogenase variant that exhibits N2 reduction that isresistant to inhibition by acetylene.232,233

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, Utah State University , 0300 Old Main Hill, Logan, Utah 84322, United States.

ABSTRACT

Two major pointscan be made regarding intermediates trapped: (1)Characterization of the E4 “Janus” intermediateas bearing four reducing equivalents in the form of two [Fe–H–Fe]bridging hydrides has provided the foundation for proposals that theFeMo-co core is never oxidized or reduced by more than one equivalentrelative to the resting-state, and that the oxidative couple in factis operative, Figure 19, I. (2) The characterizationof the common intermediates H and I, trappedduring turnover with nitrogenous substrates, led to the proposed unificationof kinetic scheme and A reaction pathway, Figure 12.

Reductive eliminationof two hydrides upon N2 binding (re mechanism) providesan explanation for the nitrogenase stoichiometry (eq 1) and for the obligatory formation of H2 upon N2 binding. This mechanism for H2 production uponN2 binding to E4, Figure 13, lower, satisfies both the stoichiometric constraint of HD formation(Chart 1, line i) and the “T+” constraint against exchange of gas-derived hydrons withsolvent (Chart 1, line ii), whereas the hpmechanism (Figure 13, upper) satisfies neither.The re mechanism further involves D2 binding to a stateat the “diazene level” of reduction, as required bythe constraint of eq 3 and Chart 1, line iii. Finally, to the best of our knowledge, all otherconstraints on the mechanism, most of which are not directly tiedto D2 binding, are satisfied, as well.

This mechanismanswers the following long-standing and oft-repeated question: Whydoes nature “waste” four ATP/two reducing equivalentsthrough an obligatory loss of H2 when N2 binds?The answer follows: reductive elimination of H2 upon bindingof N2 to FeMo-co of the E4 state generates astate in which highly reduced FeMo-co binds N2, which likelyis activated for reduction through electrostatic interactions withthe remaining two sulfur-bound protons. Transfer of the two reducingequivalents generated by the reductive elimination, combined withtransfer of the two activating protons, then forms N2H2, Figure 13, lower, in keeping withthe P–A scheme of Figure 12. It appearsthat only through this activation is the enzyme able to hydrogenateN2.

This mechanism has been supported by a rigorous test which providedexperiments in which C2H2 is added to an N2/D2 reaction mixture. Although diatomic D2 does not reduce nitrogenase C2H2 in the absenceof N2, the re mechanism successfully predicted that turnoverunder C2H2/D2/N2 wouldbreach the separation of gaseous D2 from solvent protonsby generating both C2H3D andC2H2D2.

The conclusions regardingH2 formation upon N2 binding reached from thisstudy are as follows. (i) The unprecedented incorporation of D fromD2 into the nitrogenase reduction products C2H2D2 and C2H3D duringturnover under C2H2/D2/N2 in H2O demonstrates the presence of the E4(2D) and E2(D) states under these conditions. In our viewany model that fails to incorporate obligatory H2 lossas a fundamental aspect of N2 activation is unlikely toprovide a robust description of the chemistry associated with thebiological process.242 (ii) This incorporationprovides a very clear demonstration of the essential mechanistic rolefor obligatory, reversible loss of H2 upon N2 binding and thus of the eight-electron stoichiometry for nitrogenfixation by nitrogenase embodied in eq 1. Untilnow, the data indicating that some H2 must be evolved duringN2 reduction has been viewed as being much more compellingthan the data indicating an obligatory evolution of one H2 for every N2 reduced, leading to the stoichiometry ofeq 1.17 (iii) Theformation of E4(2D) and E2(D) during turnoverunder D2/N2 in H2O is predicted bythe re mechanism for the activation of FeMo-cofactor for reductionof N2, and the interception of these intermediates by C2H2 thus provides direct experimental evidence insupport of this mechanism (Figure 17). (iv)The well-known reduction of protons by D2 to form 2HD duringturnover under D2/N2 in H2O and thenewly discovered reductions of C2H2 by D2/N2 should be viewed as being catalyzed by nitrogenasewith N2 as cocatalyst. (v) This review has proposed anexplanation of the inability of H2/D2 to reducenitrogenase and/or catalyze substrate reduction in the absence ofN2.

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