that being said, there remain a number of challenges ahead. The molecular details of
the key steps of P- and M-cluster assembly are thus far missing. X-ray structures
that capture snapshots of the key processes, such as the fusion of the P*-cluster or
the K-cluster, will therefore be instrumental. Another formidable task is to understand the role of the belt sulfur in catalysis, and to potentially connect the structural
observations with the models founded on biochemical and spectroscopic findings.
Future attempts to address these questions will without a doubt yield many more
exciting, and perhaps surprising, findings on this enzymatic system that has captivated scientist for many decades.
Acknowledgements We would also like to thank the funding agencies that support the work in
our groups, including the NIH-NIGMS grant GM67626 (to M.W.R. and Y.H.), which funds
research related to the assembly of nitrogenase; the Department of Energy grants DOE
(BES) DE-SC0016510 (to Y.H. and M.W.R.) and DE-SC0014470 (to M.W.R. and Y.H.), which
fund work related to the mechanistic investigation of ammonia and hydrocarbon formation,
respectively, by nitrogenase and related variants; and the NSF grants CHE-1904131 (to M.W.R.
and Y.H.) and CHE-1651398 (to Y.H.), which fund work related to CO and CO 2 activation by
nitrogenase and its Fe protein component, respectively.
References
1. Vitousek PM, Hättenschwiler S, Olander L, Allison S (2002) Nitrogen and nature. Ambio 31
(2):97–101
2. Gruber N, Galloway JN (2008) An Earth-system perspective of the global nitrogen cycle.
Nature 451(7176):293–296
3. Galloway JN, Cowling EB (2002) Reactive nitrogen and the world: 200 years of change.
Ambio 31(2):64–71
4. Burgess BK, Lowe DJ (1996) Mechanism of molybdenum nitrogenase. Chem Rev 96
(7):2983–3012
5. Howard JB, Rees DC (1996) Structural basis of biological nitrogen fixation. Chem Rev 96
(7):2965–2982
6. Jasniewski AJ, Lee CC, Ribbe MW, Hu Y (2020) Reactivity, mechanism, and assembly of
the alternative nitrogenases. Chem Rev. https://doi.org/10.1021/acs.chemrev.9b00704
7. Burén S, Jiménez-Vicente E, Echavarri-Erasun C, Rubio LM (2020) Biosynthesis of
nitrogenase cofactors. Chem Rev. https://doi.org/10.1021/acs.chemrev.9b00489
8. Rutledge HL, Tezcan FA (2020) Electron transfer in nitrogenase. Chem Rev. https://doi.org/
10.1021/acs.chemrev.9b00663
9. Seefeldt LC, Yang ZY, Lukoyanov DA et al (2020) Reduction of substrates by nitrogenases.
Chem Rev. https://doi.org/10.1021/acs.chemrev.9b00556
10. Rees DC (2002) Great metalloclusters in enzymology. Annu Rev Biochem 71:221–246
11. Howard JB, Rees DC (1994) Nitrogenase: a nucleotide-dependent molecular switch. Annu
Rev Biochem 63:235–264
12. Thorneley R, Lowe D (1996) Nitrogenase: substrate binding and activation. J Biol Inorg
Chem 1:576–580
13. Lindahl PA, Day EP, Kent TA, Orme-Johnson WH, Münck E (1985) Mössbauer, EPR, and
magnetization studies of the Azotobacter vinelandii Fe protein. Evidence for a [4Fe-4S]
1+
cluster with spin S = 3/2. J Biol Chem 260(20):11160–11173
14. Watt GD, McDonald JW (1985) Electron paramagnetic resonance spectrum of the iron
protein of nitrogenase: existence of a g = 4 spectral component and its effect on spin
quantization Biochemistry 24:7226–7231
Assembly and Function of Nitrogenase
179
the key steps of P- and M-cluster assembly are thus far missing. X-ray structures
that capture snapshots of the key processes, such as the fusion of the P*-cluster or
the K-cluster, will therefore be instrumental. Another formidable task is to understand the role of the belt sulfur in catalysis, and to potentially connect the structural
observations with the models founded on biochemical and spectroscopic findings.
Future attempts to address these questions will without a doubt yield many more
exciting, and perhaps surprising, findings on this enzymatic system that has captivated scientist for many decades.
Acknowledgements We would also like to thank the funding agencies that support the work in
our groups, including the NIH-NIGMS grant GM67626 (to M.W.R. and Y.H.), which funds
research related to the assembly of nitrogenase; the Department of Energy grants DOE
(BES) DE-SC0016510 (to Y.H. and M.W.R.) and DE-SC0014470 (to M.W.R. and Y.H.), which
fund work related to the mechanistic investigation of ammonia and hydrocarbon formation,
respectively, by nitrogenase and related variants; and the NSF grants CHE-1904131 (to M.W.R.
and Y.H.) and CHE-1651398 (to Y.H.), which fund work related to CO and CO 2 activation by
nitrogenase and its Fe protein component, respectively.
References
1. Vitousek PM, Hättenschwiler S, Olander L, Allison S (2002) Nitrogen and nature. Ambio 31
(2):97–101
2. Gruber N, Galloway JN (2008) An Earth-system perspective of the global nitrogen cycle.
Nature 451(7176):293–296
3. Galloway JN, Cowling EB (2002) Reactive nitrogen and the world: 200 years of change.
Ambio 31(2):64–71
4. Burgess BK, Lowe DJ (1996) Mechanism of molybdenum nitrogenase. Chem Rev 96
(7):2983–3012
5. Howard JB, Rees DC (1996) Structural basis of biological nitrogen fixation. Chem Rev 96
(7):2965–2982
6. Jasniewski AJ, Lee CC, Ribbe MW, Hu Y (2020) Reactivity, mechanism, and assembly of
the alternative nitrogenases. Chem Rev. https://doi.org/10.1021/acs.chemrev.9b00704
7. Burén S, Jiménez-Vicente E, Echavarri-Erasun C, Rubio LM (2020) Biosynthesis of
nitrogenase cofactors. Chem Rev. https://doi.org/10.1021/acs.chemrev.9b00489
8. Rutledge HL, Tezcan FA (2020) Electron transfer in nitrogenase. Chem Rev. https://doi.org/
10.1021/acs.chemrev.9b00663
9. Seefeldt LC, Yang ZY, Lukoyanov DA et al (2020) Reduction of substrates by nitrogenases.
Chem Rev. https://doi.org/10.1021/acs.chemrev.9b00556
10. Rees DC (2002) Great metalloclusters in enzymology. Annu Rev Biochem 71:221–246
11. Howard JB, Rees DC (1994) Nitrogenase: a nucleotide-dependent molecular switch. Annu
Rev Biochem 63:235–264
12. Thorneley R, Lowe D (1996) Nitrogenase: substrate binding and activation. J Biol Inorg
Chem 1:576–580
13. Lindahl PA, Day EP, Kent TA, Orme-Johnson WH, Münck E (1985) Mössbauer, EPR, and
magnetization studies of the Azotobacter vinelandii Fe protein. Evidence for a [4Fe-4S]
1+
cluster with spin S = 3/2. J Biol Chem 260(20):11160–11173
14. Watt GD, McDonald JW (1985) Electron paramagnetic resonance spectrum of the iron
protein of nitrogenase: existence of a g = 4 spectral component and its effect on spin
quantization Biochemistry 24:7226–7231
Assembly and Function of Nitrogenase
179