35. Sickerman NS, Rettberg LA, Lee CC, Hu Y, Ribbe MW (2017) Cluster assembly in
nitrogenase. Essays Biochem 61(2):271–279
36. Hu Y, Ribbe MW (2016a) Biosynthesis of the metalloclusters of nitrogenases. Annu Rev
Biochem 85:455–483
37. Ribbe MW, Hu Y, Hodgson KO, Hedman B (2014) Biosynthesis of nitrogenase
metalloclusters. Chem Rev 114(8):4063–4080
38. Smith AD, Jameson GN, Dos Santos PC et al (2005) NifS-mediated assembly of [4Fe-4S]
clusters in the N- and C-terminal domains of the NifU scaffold protein. Biochemistry 44
(39):12955–12969
39. Yuvaniyama P, Agar JN, Cash VL, Johnson MK, Dean DR (2000) NifS-directed assembly
of a transient [2Fe-2S] cluster within the NifU protein. Proc Natl Acad Sci U S A 97(2):599–
604
40. Kennedy C, Dean D (1992) The nifU, nifS and nifV gene products are required for activity
of all three nitrogenases of Azotobacter vinelandii. Mol Gen Genet 231(3):494–498
41. Ribbe MW, Hu Y, Guo M, Schmid B, Burgess BK (2002) The FeMoco-deficient MoFe
protein produced by a nifH deletion strain of Azotobacter vinelandii shows unusual P-cluster
features. J Biol Chem 277(26):23469–23476
42. Hu Y, Fay AW, Dos Santos PC, Naderi F, Ribbe MW (2004) Characterization of
Azotobacter vinelandii nifZ deletion strains. Indication of stepwise MoFe protein assembly.
J Biol Chem 279(52):54963–54971
43. Corbett MC, Hu Y, Naderi F, Ribbe MW, Hedman B, Hodgson KO (2004) Comparison of
iron-molybdenum cofactor-deficient nitrogenase MoFe proteins by X-ray absorption
spectroscopy: implications for P-cluster biosynthesis. J Biol Chem 279(27):28276–28282
44. Broach RB, Rupnik K, Hu Y et al (2006) Variable-temperature, variable-field magnetic
circular dichroism spectroscopic study of the metal clusters in the DnifB and DnifH MoFe
proteins of nitrogenase from Azotobacter vinelandii. Biochemistry 45(50):15039–15048
45. Rupnik K, Lee CC, Hu Y, Ribbe MW, Hales BJ (2011) [4Fe4S]
2+ clusters exhibit
ground-state paramagnetism. J Am Chem Soc 133(18):6871–6873
46. Lee CC, Blank MA, Fay AW et al (2009) Stepwise formation of P-cluster in nitrogenase
MoFe protein. Proc Natl Acad Sci U S A 106(44):18474–18478
47. Hu Y, Fay AW, Lee CC, Ribbe MW (2007) P-cluster maturation on nitrogenase MoFe
protein. Proc Natl Acad Sci U S A 104(25):10424–10429
48. Rupnik K, Lee CC, Hu Y, Ribbe MW, Hales BJ (2018) A VTVH MCD and EPR
spectroscopic study of the maturation of the “second” nitrogenase P-cluster. Inorg Chem 57
(8):4719–4725
49. Rupnik K, Lee CC, Wiig JA, Hu Y, Ribbe MW, Hales BJ (2014) Nonenzymatic synthesis of
the P-cluster in the nitrogenase MoFe protein: evidence of the involvement of all-ferrous
[Fe 4 S 4 ]
0 intermediates. Biochemistry 53(7):1108–1116
50. Corbett MC, Hu Y, Fay AW et al (2007) Conformational differences between Azotobacter
vinelandii nitrogenase MoFe proteins as studied by small-angle X-ray scattering. Biochemistry 46(27):8066–8074
51. Wiig JA, Hu Y, Ribbe MW (2011) NifEN-B complex of Azotobacter vinelandii is fully
functional in nitrogenase FeMo cofactor assembly. Proc Natl Acad Sci U S A 108(21):8623–
8627
52. Hu Y, Ribbe MW (2016b) Maturation of nitrogenase cofactor-the role of a class E radical
SAM methyltransferase NifB. Curr Opin Chem Biol 31:188–194
53. Frey PA, Hegeman AD, Ruzicka FJ (2008) The radical SAM superfamily. Crit Rev Biochem
Mol Biol 43(1):63–88
54. Wiig JA, Hu Y, Lee CC, Ribbe MW (2012) Radical SAM-dependent carbon insertion into
the nitrogenase M-cluster. Science 337(6102):1672–1675
55. Wiig JA, Hu Y, Ribbe MW (2015) Refining the pathway of carbide insertion into the
nitrogenase M-cluster. Nat Commun 6:8034
Assembly and Function of Nitrogenase
181
nitrogenase. Essays Biochem 61(2):271–279
36. Hu Y, Ribbe MW (2016a) Biosynthesis of the metalloclusters of nitrogenases. Annu Rev
Biochem 85:455–483
37. Ribbe MW, Hu Y, Hodgson KO, Hedman B (2014) Biosynthesis of nitrogenase
metalloclusters. Chem Rev 114(8):4063–4080
38. Smith AD, Jameson GN, Dos Santos PC et al (2005) NifS-mediated assembly of [4Fe-4S]
clusters in the N- and C-terminal domains of the NifU scaffold protein. Biochemistry 44
(39):12955–12969
39. Yuvaniyama P, Agar JN, Cash VL, Johnson MK, Dean DR (2000) NifS-directed assembly
of a transient [2Fe-2S] cluster within the NifU protein. Proc Natl Acad Sci U S A 97(2):599–
604
40. Kennedy C, Dean D (1992) The nifU, nifS and nifV gene products are required for activity
of all three nitrogenases of Azotobacter vinelandii. Mol Gen Genet 231(3):494–498
41. Ribbe MW, Hu Y, Guo M, Schmid B, Burgess BK (2002) The FeMoco-deficient MoFe
protein produced by a nifH deletion strain of Azotobacter vinelandii shows unusual P-cluster
features. J Biol Chem 277(26):23469–23476
42. Hu Y, Fay AW, Dos Santos PC, Naderi F, Ribbe MW (2004) Characterization of
Azotobacter vinelandii nifZ deletion strains. Indication of stepwise MoFe protein assembly.
J Biol Chem 279(52):54963–54971
43. Corbett MC, Hu Y, Naderi F, Ribbe MW, Hedman B, Hodgson KO (2004) Comparison of
iron-molybdenum cofactor-deficient nitrogenase MoFe proteins by X-ray absorption
spectroscopy: implications for P-cluster biosynthesis. J Biol Chem 279(27):28276–28282
44. Broach RB, Rupnik K, Hu Y et al (2006) Variable-temperature, variable-field magnetic
circular dichroism spectroscopic study of the metal clusters in the DnifB and DnifH MoFe
proteins of nitrogenase from Azotobacter vinelandii. Biochemistry 45(50):15039–15048
45. Rupnik K, Lee CC, Hu Y, Ribbe MW, Hales BJ (2011) [4Fe4S]
2+ clusters exhibit
ground-state paramagnetism. J Am Chem Soc 133(18):6871–6873
46. Lee CC, Blank MA, Fay AW et al (2009) Stepwise formation of P-cluster in nitrogenase
MoFe protein. Proc Natl Acad Sci U S A 106(44):18474–18478
47. Hu Y, Fay AW, Lee CC, Ribbe MW (2007) P-cluster maturation on nitrogenase MoFe
protein. Proc Natl Acad Sci U S A 104(25):10424–10429
48. Rupnik K, Lee CC, Hu Y, Ribbe MW, Hales BJ (2018) A VTVH MCD and EPR
spectroscopic study of the maturation of the “second” nitrogenase P-cluster. Inorg Chem 57
(8):4719–4725
49. Rupnik K, Lee CC, Wiig JA, Hu Y, Ribbe MW, Hales BJ (2014) Nonenzymatic synthesis of
the P-cluster in the nitrogenase MoFe protein: evidence of the involvement of all-ferrous
[Fe 4 S 4 ]
0 intermediates. Biochemistry 53(7):1108–1116
50. Corbett MC, Hu Y, Fay AW et al (2007) Conformational differences between Azotobacter
vinelandii nitrogenase MoFe proteins as studied by small-angle X-ray scattering. Biochemistry 46(27):8066–8074
51. Wiig JA, Hu Y, Ribbe MW (2011) NifEN-B complex of Azotobacter vinelandii is fully
functional in nitrogenase FeMo cofactor assembly. Proc Natl Acad Sci U S A 108(21):8623–
8627
52. Hu Y, Ribbe MW (2016b) Maturation of nitrogenase cofactor-the role of a class E radical
SAM methyltransferase NifB. Curr Opin Chem Biol 31:188–194
53. Frey PA, Hegeman AD, Ruzicka FJ (2008) The radical SAM superfamily. Crit Rev Biochem
Mol Biol 43(1):63–88
54. Wiig JA, Hu Y, Lee CC, Ribbe MW (2012) Radical SAM-dependent carbon insertion into
the nitrogenase M-cluster. Science 337(6102):1672–1675
55. Wiig JA, Hu Y, Ribbe MW (2015) Refining the pathway of carbide insertion into the
nitrogenase M-cluster. Nat Commun 6:8034
Assembly and Function of Nitrogenase
181