α-Lys Participates in Insertion of FeMoco to MoFe Protein and Maintains Nitrogenase Activity in M5al

Overview

Journal Front Microbiol

Specialty Microbiology

Date 2019 May 7

PMID 31057512

Authors

Lina Song

Pengxi Liu

Wei Jiang

Qingjuan Guo

Chunxi Zhang

Abdul Basit

Ying Li

Jilun Li

Affiliations

Soon will be listed here.

Abstract

Our previous investigation of substrates reduction catalyzed by nitrogenase suggested that α-Ile of MoFe protein possibly functions as an electron transfer gate to Mo site of active center-"FeMoco". Amino acid residue α-Lys connects directly to α-Ile, and they are located in the same α-helix (α423-431). In the present study, function of α-Lys was investigated by replacing it with Arg (alkaline, like Lys), Gln (neutral), Glu (acidic), and Ala (neutral) through site-directed mutagenesis and homologous recombination. The mutants were, respectively, termed 424R, 424Q, 424E, and 424A. Studies of diazotrophic cell growth, cytological, and enzymatic properties indicated that none of the substitutions altered the secondary structure of MoFe protein, or normal expression of , , and . Substitution of alkaline amino acid (i.e., 424R) maintained acetylene (CH) and proton (H) reduction activities at normal levels similar to that of wild-type (WT), because its FeMoco content did not reduce. In contrast, substitution of acidic or neutral amino acid (i.e., 424Q, 424E, 424A) impaired the catalytic activity of nitrogenase to varying degrees. Combination of MoFe protein structural simulation and the results of a series of experiments, the function of α-Lys in ensuring insertion of FeMoco to MoFe protein was further confirmed, and the contribution of α-Lys in maintaining low potential of the microenvironment causing efficient catalytic activity of nitrogenase was demonstrated.

References

Wang S, Jin W, Chen H, Zhou Z . Comparison of hydroxycarboxylato imidazole molybdenum(iv) complexes and nitrogenase protein structures: indirect evidence for the protonation of homocitrato FeMo-cofactors. Dalton Trans. 2018; 47(22):7412-7421. DOI: 10.1039/c8dt00278a. View

Kim C, Newton W, Dean D . Role of the MoFe protein alpha-subunit histidine-195 residue in FeMo-cofactor binding and nitrogenase catalysis. Biochemistry. 1995; 34(9):2798-808. DOI: 10.1021/bi00009a008. View

Lancaster K, Roemelt M, Ettenhuber P, Hu Y, Ribbe M, Neese F . X-ray emission spectroscopy evidences a central carbon in the nitrogenase iron-molybdenum cofactor. Science. 2011; 334(6058):974-7. PMC: 3800678. DOI: 10.1126/science.1206445. View

Yang J, Xie X, Yang M, Dixon R, Wang Y . Modular electron-transport chains from eukaryotic organelles function to support nitrogenase activity. Proc Natl Acad Sci U S A. 2017; 114(12):E2460-E2465. PMC: 5373397. DOI: 10.1073/pnas.1620058114. View

Hoffman B, Lukoyanov D, Yang Z, Dean D, Seefeldt L . Mechanism of nitrogen fixation by nitrogenase: the next stage. Chem Rev. 2014; 114(8):4041-62. PMC: 4012840. DOI: 10.1021/cr400641x. View

Liang J, BURRIS R . Hydrogen burst associated with nitrogenase-catalyzed reactions. Proc Natl Acad Sci U S A. 1988; 85(24):9446-50. PMC: 282769. DOI: 10.1073/pnas.85.24.9446. View

Allen R, Tilbrook K, Warden A, Campbell P, Rolland V, Singh S . Expression of 16 Nitrogenase Proteins within the Plant Mitochondrial Matrix. Front Plant Sci. 2017; 8:287. PMC: 5334340. DOI: 10.3389/fpls.2017.00287. View

Liang J, Madden M, Shah V, BURRIS R . Citrate substitutes for homocitrate in nitrogenase of a nifV mutant of Klebsiella pneumoniae. Biochemistry. 1990; 29(37):8577-81. DOI: 10.1021/bi00489a011. View

Simpson F, BURRIS R . A nitrogen pressure of 50 atmospheres does not prevent evolution of hydrogen by nitrogenase. Science. 1984; 224(4653):1095-7. DOI: 10.1126/science.6585956. View

10.

Spatzal T, Aksoyoglu M, Zhang L, Andrade S, Schleicher E, Weber S . Evidence for interstitial carbon in nitrogenase FeMo cofactor. Science. 2011; 334(6058):940. PMC: 3268367. DOI: 10.1126/science.1214025. View

11.

Eady R, Robson R, Richardson T, Miller R, Hawkins M . The vanadium nitrogenase of Azotobacter chroococcum. Purification and properties of the VFe protein. Biochem J. 1987; 244(1):197-207. PMC: 1147972. DOI: 10.1042/bj2440197. View

12.

Danyal K, Dean D, Hoffman B, Seefeldt L . Electron transfer within nitrogenase: evidence for a deficit-spending mechanism. Biochemistry. 2011; 50(43):9255-63. PMC: 3202676. DOI: 10.1021/bi201003a. View

13.

Durrant M, Francis A, Lowe D, Newton W, Fisher K . Evidence for a dynamic role for homocitrate during nitrogen fixation: the effect of substitution at the alpha-Lys426 position in MoFe-protein of Azotobacter vinelandii. Biochem J. 2006; 397(2):261-70. PMC: 1513279. DOI: 10.1042/BJ20060102. View

14.

Burgess B, Lowe D . Mechanism of Molybdenum Nitrogenase. Chem Rev. 1996; 96(7):2983-3012. DOI: 10.1021/cr950055x. View

15.

Li J, BURRIS R . Influence of pN2 and pD2 on HD formation by various nitrogenases. Biochemistry. 1983; 22(19):4472-80. DOI: 10.1021/bi00288a019. View

16.

Kaczmarek M, Malhotra A, Balan G, Timmins A, de Visser S . Nitrogen Reduction to Ammonia on a Biomimetic Mononuclear Iron Centre: Insights into the Nitrogenase Enzyme. Chemistry. 2017; 24(20):5293-5302. PMC: 5915742. DOI: 10.1002/chem.201704688. View

17.

Buren S, Jiang X, Lopez-Torrejon G, Echavarri-Erasun C, Rubio L . Purification and Activity of Mitochondria Targeted Nitrogenase Cofactor Maturase NifB. Front Plant Sci. 2017; 8:1567. PMC: 5601070. DOI: 10.3389/fpls.2017.01567. View

18.

Schmid B, Ribbe M, Einsle O, Yoshida M, Thomas L, Dean D . Structure of a cofactor-deficient nitrogenase MoFe protein. Science. 2002; 296(5566):352-6. DOI: 10.1126/science.1070010. View

19.

Bulen W, LECOMTE J . The nitrogenase system from Azotobacter: two-enzyme requirement for N2 reduction, ATP-dependent H2 evolution, and ATP hydrolysis. Proc Natl Acad Sci U S A. 1966; 56(3):979-86. PMC: 219956. DOI: 10.1073/pnas.56.3.979. View

20.

Georgiadis M, Komiya H, Chakrabarti P, Woo D, Kornuc J, Rees D . Crystallographic structure of the nitrogenase iron protein from Azotobacter vinelandii. Science. 1992; 257(5077):1653-9. DOI: 10.1126/science.1529353. View