Mo-, V-, and Fe-Nitrogenases Use a Universal Eight-Electron Reductive-Elimination Mechanism to Achieve N2 Reduction

Derek F. Harris, Dmitriy A. Lukoyanov, Hayden Kallas, Christian Trncik, Zhi Yong Yang, Philip D Compton, Neil Kelleher, Oliver Einsle, Dennis R. Dean, Brian M Hoffman, Lance C. Seefeldt*

*Corresponding author for this work

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Abstract

Three genetically distinct, but structurally similar, isozymes of nitrogenase catalyze biological N2 reduction to 2NH3: Mo-, V-, and Fe-nitrogenase, named respectively for the metal (M) in their active site metallocofactors (metal-ion composition, MFe7). Studies of the Mo-enzyme have revealed key aspects of its mechanism for N2 binding and reduction. Central to this mechanism is accumulation of four electrons and protons on its active site metallocofactor, called FeMo-co, as metal bound hydrides to generate the key E4(4H) ("Janus") state. N2 binding/reduction in this state is coupled to reductive elimination (re) of the two hydrides as H2, the forward direction of a reductive-elimination/oxidative-addition (re/oa) equilibrium. A recent study demonstrated that Fe-nitrogenase follows the same re/oa mechanism, as particularly evidenced by HD formation during turnover under N2/D2. Kinetic analysis revealed that Mo- and Fe-nitrogenases show similar rate constants for hydrogenase-like H2 formation by hydride protonolysis (kHP) but significant differences in the rate constant for H2 re with N2 binding/reduction (kre). We now report that V-nitrogenase also exhibits HD formation during N2/D2 turnover (and H2 inhibition of N2 reduction), thereby establishing the re/oa equilibrium as a universal mechanism for N2 binding and activation among the three nitrogenases. Kinetic analysis further reveals that differences in catalytic efficiencies do not stem from significant differences in the rate constant (kHP) for H2 production by the hydrogenase-like side reaction but directly arise from the differences in the rate constant (kre) for the re of H2 coupled to N2 binding/reduction, which decreases in the order Mo > V > Fe.

Original languageEnglish (US)
Pages (from-to)3293-3301
Number of pages9
JournalBiochemistry
Volume58
Issue number30
DOIs
StatePublished - Jul 30 2019

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Nitrogenase
Electrons
Rate constants
Hydrides
Hydrogenase
Metals
Catalytic Domain
Kinetics
Isoenzymes
Metal ions
Protons
Chemical activation
Ions
Enzymes
Chemical analysis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Harris, Derek F. ; Lukoyanov, Dmitriy A. ; Kallas, Hayden ; Trncik, Christian ; Yang, Zhi Yong ; Compton, Philip D ; Kelleher, Neil ; Einsle, Oliver ; Dean, Dennis R. ; Hoffman, Brian M ; Seefeldt, Lance C. / Mo-, V-, and Fe-Nitrogenases Use a Universal Eight-Electron Reductive-Elimination Mechanism to Achieve N2 Reduction. In: Biochemistry. 2019 ; Vol. 58, No. 30. pp. 3293-3301.
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title = "Mo-, V-, and Fe-Nitrogenases Use a Universal Eight-Electron Reductive-Elimination Mechanism to Achieve N2 Reduction",
abstract = "Three genetically distinct, but structurally similar, isozymes of nitrogenase catalyze biological N2 reduction to 2NH3: Mo-, V-, and Fe-nitrogenase, named respectively for the metal (M) in their active site metallocofactors (metal-ion composition, MFe7). Studies of the Mo-enzyme have revealed key aspects of its mechanism for N2 binding and reduction. Central to this mechanism is accumulation of four electrons and protons on its active site metallocofactor, called FeMo-co, as metal bound hydrides to generate the key E4(4H) ({"}Janus{"}) state. N2 binding/reduction in this state is coupled to reductive elimination (re) of the two hydrides as H2, the forward direction of a reductive-elimination/oxidative-addition (re/oa) equilibrium. A recent study demonstrated that Fe-nitrogenase follows the same re/oa mechanism, as particularly evidenced by HD formation during turnover under N2/D2. Kinetic analysis revealed that Mo- and Fe-nitrogenases show similar rate constants for hydrogenase-like H2 formation by hydride protonolysis (kHP) but significant differences in the rate constant for H2 re with N2 binding/reduction (kre). We now report that V-nitrogenase also exhibits HD formation during N2/D2 turnover (and H2 inhibition of N2 reduction), thereby establishing the re/oa equilibrium as a universal mechanism for N2 binding and activation among the three nitrogenases. Kinetic analysis further reveals that differences in catalytic efficiencies do not stem from significant differences in the rate constant (kHP) for H2 production by the hydrogenase-like side reaction but directly arise from the differences in the rate constant (kre) for the re of H2 coupled to N2 binding/reduction, which decreases in the order Mo > V > Fe.",
author = "Harris, {Derek F.} and Lukoyanov, {Dmitriy A.} and Hayden Kallas and Christian Trncik and Yang, {Zhi Yong} and Compton, {Philip D} and Neil Kelleher and Oliver Einsle and Dean, {Dennis R.} and Hoffman, {Brian M} and Seefeldt, {Lance C.}",
year = "2019",
month = "7",
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doi = "10.1021/acs.biochem.9b00468",
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Harris, DF, Lukoyanov, DA, Kallas, H, Trncik, C, Yang, ZY, Compton, PD, Kelleher, N, Einsle, O, Dean, DR, Hoffman, BM & Seefeldt, LC 2019, 'Mo-, V-, and Fe-Nitrogenases Use a Universal Eight-Electron Reductive-Elimination Mechanism to Achieve N2 Reduction', Biochemistry, vol. 58, no. 30, pp. 3293-3301. https://doi.org/10.1021/acs.biochem.9b00468

Mo-, V-, and Fe-Nitrogenases Use a Universal Eight-Electron Reductive-Elimination Mechanism to Achieve N2 Reduction. / Harris, Derek F.; Lukoyanov, Dmitriy A.; Kallas, Hayden; Trncik, Christian; Yang, Zhi Yong; Compton, Philip D; Kelleher, Neil; Einsle, Oliver; Dean, Dennis R.; Hoffman, Brian M; Seefeldt, Lance C.

In: Biochemistry, Vol. 58, No. 30, 30.07.2019, p. 3293-3301.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Mo-, V-, and Fe-Nitrogenases Use a Universal Eight-Electron Reductive-Elimination Mechanism to Achieve N2 Reduction

AU - Harris, Derek F.

AU - Lukoyanov, Dmitriy A.

AU - Kallas, Hayden

AU - Trncik, Christian

AU - Yang, Zhi Yong

AU - Compton, Philip D

AU - Kelleher, Neil

AU - Einsle, Oliver

AU - Dean, Dennis R.

AU - Hoffman, Brian M

AU - Seefeldt, Lance C.

PY - 2019/7/30

Y1 - 2019/7/30

N2 - Three genetically distinct, but structurally similar, isozymes of nitrogenase catalyze biological N2 reduction to 2NH3: Mo-, V-, and Fe-nitrogenase, named respectively for the metal (M) in their active site metallocofactors (metal-ion composition, MFe7). Studies of the Mo-enzyme have revealed key aspects of its mechanism for N2 binding and reduction. Central to this mechanism is accumulation of four electrons and protons on its active site metallocofactor, called FeMo-co, as metal bound hydrides to generate the key E4(4H) ("Janus") state. N2 binding/reduction in this state is coupled to reductive elimination (re) of the two hydrides as H2, the forward direction of a reductive-elimination/oxidative-addition (re/oa) equilibrium. A recent study demonstrated that Fe-nitrogenase follows the same re/oa mechanism, as particularly evidenced by HD formation during turnover under N2/D2. Kinetic analysis revealed that Mo- and Fe-nitrogenases show similar rate constants for hydrogenase-like H2 formation by hydride protonolysis (kHP) but significant differences in the rate constant for H2 re with N2 binding/reduction (kre). We now report that V-nitrogenase also exhibits HD formation during N2/D2 turnover (and H2 inhibition of N2 reduction), thereby establishing the re/oa equilibrium as a universal mechanism for N2 binding and activation among the three nitrogenases. Kinetic analysis further reveals that differences in catalytic efficiencies do not stem from significant differences in the rate constant (kHP) for H2 production by the hydrogenase-like side reaction but directly arise from the differences in the rate constant (kre) for the re of H2 coupled to N2 binding/reduction, which decreases in the order Mo > V > Fe.

AB - Three genetically distinct, but structurally similar, isozymes of nitrogenase catalyze biological N2 reduction to 2NH3: Mo-, V-, and Fe-nitrogenase, named respectively for the metal (M) in their active site metallocofactors (metal-ion composition, MFe7). Studies of the Mo-enzyme have revealed key aspects of its mechanism for N2 binding and reduction. Central to this mechanism is accumulation of four electrons and protons on its active site metallocofactor, called FeMo-co, as metal bound hydrides to generate the key E4(4H) ("Janus") state. N2 binding/reduction in this state is coupled to reductive elimination (re) of the two hydrides as H2, the forward direction of a reductive-elimination/oxidative-addition (re/oa) equilibrium. A recent study demonstrated that Fe-nitrogenase follows the same re/oa mechanism, as particularly evidenced by HD formation during turnover under N2/D2. Kinetic analysis revealed that Mo- and Fe-nitrogenases show similar rate constants for hydrogenase-like H2 formation by hydride protonolysis (kHP) but significant differences in the rate constant for H2 re with N2 binding/reduction (kre). We now report that V-nitrogenase also exhibits HD formation during N2/D2 turnover (and H2 inhibition of N2 reduction), thereby establishing the re/oa equilibrium as a universal mechanism for N2 binding and activation among the three nitrogenases. Kinetic analysis further reveals that differences in catalytic efficiencies do not stem from significant differences in the rate constant (kHP) for H2 production by the hydrogenase-like side reaction but directly arise from the differences in the rate constant (kre) for the re of H2 coupled to N2 binding/reduction, which decreases in the order Mo > V > Fe.

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U2 - 10.1021/acs.biochem.9b00468

DO - 10.1021/acs.biochem.9b00468

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