76. Fay AW, Blank MA, Yoshizawa JM et al (2010) Formation of a homocitrate-free
iron-molybdenum cluster on NifEN: implications for the role of homocitrate in nitrogenase
assembly. Dalton Trans 39(12):3124–3130
77. Solomon JB, Lee CC, Jasniewski AJ, Rasekh MF, Ribbe MW, Hu Y (2020) Heterologous
expression and engineering of the nitrogenase cofactor biosynthesis scaffold NifEN. Angew
Chem Int Ed Engl 59(17):6887–6893
78. Hu Y, Fay AW, Ribbe MW (2007) Molecular insights into nitrogenase FeMo cofactor
insertion: the role of His 362 of the MoFe protein alpha subunit in FeMo cofactor
incorporation. J Biol Inorg Chem 12(4):449–460
79. Fay AW, Hu Y, Schmid B, Ribbe MW (2007) Molecular insights into nitrogenase FeMoco
insertion–the role of His 274 and His 451 of MoFe protein alpha subunit. J Inorg Biochem
101(11–12):1630–1641
80. Hu Y, Fay AW, Schmid B, Makar B, Ribbe MW (2006) Molecular insights into nitrogenase
FeMoco insertion: TRP-444 of MoFe protein alpha-subunit locks FeMoco in its binding site.
J Biol Chem 281(41):30534–30541
81. Thorneley RN, Lowe DJ (1984) The mechanism of Klebsiella pneumoniae nitrogenase
action. Pre-steady-state kinetics of an enzyme-bound intermediate in N 2 reduction and of
NH 3 formation. Biochem J 224(3):887–894
82. Hoffman BM, Lukoyanov D, Yang ZY, Dean DR, Seefeldt LC (2014) Mechanism of
nitrogen fixation by nitrogenase: the next stage. Chem Rev 114(8):4041–4062
83. Lowe DJ, Thorneley RN (1984) The mechanism of Klebsiella pneumoniae nitrogenase
action. The determination of rate constants required for the simulation of the kinetics of N 2
reduction and H 2 evolution. Biochem J 224(3):895–901
84. Thorneley RN, Lowe DJ (1984) The mechanism of Klebsiella pneumoniae nitrogenase
action. Simulation of the dependences of H 2 -evolution rate on component-protein
concentration and ratio and sodium dithionite concentration. Biochem J 224(3):903–909
85. Lukoyanov D, Yang ZY, Khadka N, Dean DR, Seefeldt LC, Hoffman BM (2015)
Identification of a key catalytic intermediate demonstrates that nitrogenase is activated by the
reversible exchange of N 2 for H 2 . J Am Chem Soc 137(10):3610–3615
86. Lukoyanov D, Khadka N, Yang ZY, Dean DR, Seefeldt LC, Hoffman BM (2016a)
Reversible photoinduced reductive elimination of H 2 from the nitrogenase dihydride state,
the E 4 (4H) Janus intermediate. J Am Chem Soc 138(4):1320–1327
87. Lukoyanov D, Khadka N, Yang ZY, Dean DR, Seefeldt LC, Hoffman BM (2016b)
Reductive elimination of H 2 activates nitrogenase to reduce the NN triple Bond:
characterization of the E 4 (4H) Janus intermediate in wild-type enzyme. J Am Chem Soc 138
(33):10674–10683
88. Igarashi RY, Laryukhin M, Dos Santos PC et al (2005) Trapping H- bound to the
nitrogenase FeMo-cofactor active site during H 2 evolution: characterization by ENDOR
spectroscopy. J Am Chem Soc 127(17):6231–6241
89. Lukoyanov D, Barney BM, Dean DR, Seefeldt LC, Hoffman BM (2007) Connecting
nitrogenase intermediates with the kinetic scheme for N2 reduction by a relaxation protocol
and identification of the N 2 binding state. Proc Natl Acad Sci U S A 104(5):1451–1455
90. Lukoyanov D, Yang ZY, Dean DR, Seefeldt LC, Hoffman BM (2010) Is Mo involved in
hydride binding by the four-electron reduced (E4) intermediate of the nitrogenase MoFe
protein? J Am Chem Soc 132(8):2526–2527
91. Raugei S, Seefeldt LC, Hoffman BM (2018) Critical computational analysis illuminates the
reductive-elimination mechanism that activates nitrogenase for N 2 reduction. Proc Natl Acad
Sci U S A 115(45):E10521–E10530
92. Hoeke V, Tociu L, Case DA, Seefeldt LC, Raugei S, Hoffman BM (2019) High-resolution
ENDOR spectroscopy combined with quantum chemical calculations reveals the structure of
nitrogenase Janus intermediate E 4 (4H). J Am Chem Soc 141(30):11984–11996
Assembly and Function of Nitrogenase
183
iron-molybdenum cluster on NifEN: implications for the role of homocitrate in nitrogenase
assembly. Dalton Trans 39(12):3124–3130
77. Solomon JB, Lee CC, Jasniewski AJ, Rasekh MF, Ribbe MW, Hu Y (2020) Heterologous
expression and engineering of the nitrogenase cofactor biosynthesis scaffold NifEN. Angew
Chem Int Ed Engl 59(17):6887–6893
78. Hu Y, Fay AW, Ribbe MW (2007) Molecular insights into nitrogenase FeMo cofactor
insertion: the role of His 362 of the MoFe protein alpha subunit in FeMo cofactor
incorporation. J Biol Inorg Chem 12(4):449–460
79. Fay AW, Hu Y, Schmid B, Ribbe MW (2007) Molecular insights into nitrogenase FeMoco
insertion–the role of His 274 and His 451 of MoFe protein alpha subunit. J Inorg Biochem
101(11–12):1630–1641
80. Hu Y, Fay AW, Schmid B, Makar B, Ribbe MW (2006) Molecular insights into nitrogenase
FeMoco insertion: TRP-444 of MoFe protein alpha-subunit locks FeMoco in its binding site.
J Biol Chem 281(41):30534–30541
81. Thorneley RN, Lowe DJ (1984) The mechanism of Klebsiella pneumoniae nitrogenase
action. Pre-steady-state kinetics of an enzyme-bound intermediate in N 2 reduction and of
NH 3 formation. Biochem J 224(3):887–894
82. Hoffman BM, Lukoyanov D, Yang ZY, Dean DR, Seefeldt LC (2014) Mechanism of
nitrogen fixation by nitrogenase: the next stage. Chem Rev 114(8):4041–4062
83. Lowe DJ, Thorneley RN (1984) The mechanism of Klebsiella pneumoniae nitrogenase
action. The determination of rate constants required for the simulation of the kinetics of N 2
reduction and H 2 evolution. Biochem J 224(3):895–901
84. Thorneley RN, Lowe DJ (1984) The mechanism of Klebsiella pneumoniae nitrogenase
action. Simulation of the dependences of H 2 -evolution rate on component-protein
concentration and ratio and sodium dithionite concentration. Biochem J 224(3):903–909
85. Lukoyanov D, Yang ZY, Khadka N, Dean DR, Seefeldt LC, Hoffman BM (2015)
Identification of a key catalytic intermediate demonstrates that nitrogenase is activated by the
reversible exchange of N 2 for H 2 . J Am Chem Soc 137(10):3610–3615
86. Lukoyanov D, Khadka N, Yang ZY, Dean DR, Seefeldt LC, Hoffman BM (2016a)
Reversible photoinduced reductive elimination of H 2 from the nitrogenase dihydride state,
the E 4 (4H) Janus intermediate. J Am Chem Soc 138(4):1320–1327
87. Lukoyanov D, Khadka N, Yang ZY, Dean DR, Seefeldt LC, Hoffman BM (2016b)
Reductive elimination of H 2 activates nitrogenase to reduce the NN triple Bond:
characterization of the E 4 (4H) Janus intermediate in wild-type enzyme. J Am Chem Soc 138
(33):10674–10683
88. Igarashi RY, Laryukhin M, Dos Santos PC et al (2005) Trapping H- bound to the
nitrogenase FeMo-cofactor active site during H 2 evolution: characterization by ENDOR
spectroscopy. J Am Chem Soc 127(17):6231–6241
89. Lukoyanov D, Barney BM, Dean DR, Seefeldt LC, Hoffman BM (2007) Connecting
nitrogenase intermediates with the kinetic scheme for N2 reduction by a relaxation protocol
and identification of the N 2 binding state. Proc Natl Acad Sci U S A 104(5):1451–1455
90. Lukoyanov D, Yang ZY, Dean DR, Seefeldt LC, Hoffman BM (2010) Is Mo involved in
hydride binding by the four-electron reduced (E4) intermediate of the nitrogenase MoFe
protein? J Am Chem Soc 132(8):2526–2527
91. Raugei S, Seefeldt LC, Hoffman BM (2018) Critical computational analysis illuminates the
reductive-elimination mechanism that activates nitrogenase for N 2 reduction. Proc Natl Acad
Sci U S A 115(45):E10521–E10530
92. Hoeke V, Tociu L, Case DA, Seefeldt LC, Raugei S, Hoffman BM (2019) High-resolution
ENDOR spectroscopy combined with quantum chemical calculations reveals the structure of
nitrogenase Janus intermediate E 4 (4H). J Am Chem Soc 141(30):11984–11996
Assembly and Function of Nitrogenase
183