Biochemistry of Nitrogen Fixation

Luis M. Rubio Herrero (PI)

Associate Professor
Technical University of Madrid and Center for Plant Biotechnology and Genomics, Madrid, Spain




Other group members

Elena Caro Bernat

Assistant Professor

Gema López Torrejón

Assistant Lecturer

Carlos Echevarri Erasun

Assistant Lecturer

Stefan Burén


José Antonio Aznar Moreno


Xi Jiang

PhD Student

Lucía Payá Tormo

PhD Student

Álvaro Eseverri Sabaté

PhD Student

Katarzyna Dobrzynkska

PhD Student

Joanna Natalia Soldek

PhD Student

Baltasar Rubio Herrero

Management Technician

Marcel Veldhuizen

Laboratory Technician

Diana María Coroian

Laboratory Technician

Cristina Vaca Sanz

Laboratory Technician

Álvaro Salinero Lanzarote

Laboratory Technician

Research lines

  • Nitrogenase bioengineering in plants and yeast
  • Structure-function studies on nitrogenase iron-molybdenum cofactor biosynthesis
  • Optimization of hydrogen production by nitrogenase

Representative publications

  • Eseverri Á, Baysal C, Medina V, Capell T, Christou P, Rubio LM, Caro E (2020) Transit peptides from photosynthesis-related proteins mediate import of a marker protein into different plastid types and within different s Front Plant Sci 11: 560701 (doi: 10.3389/fpls.2020.560701).
  • Fajardo AS, Legrand P, Payá-Tormo L, Martin L, Pellicer Martı́nez MT, Echavarri-Erasun C, Vernède X, Rubio LM, Nicolet Y (2020) Structural insights into the mechanism of the radical SAM carbide synthase NifB, a key nitrogenase cofactor maturating e J Am Chem Soc 142: 11006-11012.
  • Eseverri Á, López‐Torrejón G, Jiang X, Burén S, Rubio LM, Caro E (2020) Use of synthetic biology tools to optimize the production of active nitrogenase Fe protein in chloroplasts of tobacco leaf cells. Plant Biotech J 18: 1882-1896.
  • Burén S, Jiménez-Vicente E, Echavarri-Erasun C, Rubio LM (2020) Biosynthesis of Nitrogenase Cofactors. Chem Rev 120: 4921-4968.
  • Burén S, Pratt K, Jiang X, Guo Y, Jimenez-Vicente E, Echavarri-Erasun C, Dean DR, Saaem I, Gordon DB, Voigt CA, Rubio LM (2019) Biosynthesis of the nitrogenase active-site cofactor precursor NifB-co in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 116: 25078-25086.
  • Baysal C, Pérez-González A, Eseverri Á, Jiang X, Medina V, Caro E, Rubio LM, Christou P, Zhu C (2019) Recognition motifs rather than phylogenetic origin influence the ability of targeting peptides to import nuclear-encoded recombinant proteins into rice mitochondria. Transgenic Res 29: 37-52.
  • Navarro-Rodríguez M, Buesa JM, Rubio LM (2019) Genetic and biochemical analysis of the Azotobacter vinelandii molybdenum storage protein. Front Microbiol 10: 579 (doi: 10.3389/fmicb.2019.00579).
  • Burén S, López-Torrejón G, Rubio LM (2018). Extreme bioengineering to meet the nitrogen challenge. Proc Natl Acad Sci USA 115: 8849-8851.
  • Jimenez-Vicente E, Yang ZY, Ray WK, Echavarri-Erasun C, Cash VL, Rubio LM, Seefeldt LC, Dean DR (2018) Sequential and differential interaction of assembly factors during nitrogenase MoFe protein maturation. J Biol Chem 293: 9812-9823.
  • Burén S, Rubio LM (2017) State of the art in eukaryotic nitrogenase engineering. FEMS Microbiol Lett 365: 274 (doi: 10.1093/femsle/fnx274).
  • Arragain S, Jiménez-Vicente E, Scandurra AA, Burén S, Rubio LM, Echavarri-Erasun C (2017) Diversity and functional analysis of the FeMo-cofactor maturase NifB. Front Plant Sci 8: 1947 (doi: 10.3389/fpls.2017.01947).
  • Pérez-González A, Kniewel R, Veldhuizen M, Verma HK, Navarro-Rodríguez M, Rubio LM, Caro E (2017) Adaptation of the GoldenBraid modular cloning system and creation of a toolkit for the expression of heterologous proteins in yeast mitochondria. BMC Biotech 17: 80 (doi: 10.1186/s12896-017-0393-y).
  • Burén S, Jiang X, López-Torrejón G, Echavarri-Erasun C, Rubio LM (2017) Purification and in vitro activity of mitochondria targeted nitrogenase cofactor maturase NifB. Front Plant Sci 8:1567 (doi: 10.3389/fpls.2017.01567).
  • Burén S, Young EM, Sweeny EA, Lopez-Torrejón G, Veldhuizen M, Voigt CA, Rubio LM (2017) Formation of nitrogenase NifDK tetramers in the mitochondria of Saccharomyces cerevisiae. ACS Synth Biol 6: 1043-1055.


  • Rubio LM (PI) (2020-2024) BNF Cereals Phase III. Bill & Melinda Gates Foundation. INV005889.
  • Rubio LM (PI) (2018-2021) New paradigms in nitrogenase biogenesis. Ministry of Science, Innovation and Universities. BIO2017-88475-R.
  • Rubio LM (PI) (2016-2020) BNF Cereals Phase II. Bill & Melinda Gates Foundation. OPP1143172.
  • Rubio LM (PI) (2015-2017) Biotechnological applications of nitrogenase. Ministry of Economy and Competitiveness. BIO20154-59131-R.
  • Rubio LM (PI) (2011-2016) Engineering nitrogen fixation in mitochondria. Bill & Melinda Gates Foundation. OPP10442444.

Relevant methods

  • Nitrogenase activity assays
  • Anaerobic biochemistry
  • In vitro synthesis of protein metal cofactors
  • Purification and analysis of metalloproteins
  • Synthetic biology in bacteria, yeast and plants
  • Bacteria and yeast fermentations
  • Protein-protein and protein-metal interactions

Collaborations with other national and international research groups

  • Dennis Dean (Virginia Tech, USA)
  • Christopher Voigt (Massachusetts Institute of Technology, USA)
  • Leonardo Curatti (FIBA, Argentina)
  • Yvain Nicolet (Institut de Biologie Structurale, France)
  • Lance Seefeldt (Utah State, USA)
  • Yisong Guo (Carnegie Mellon University, USA)
  • Paul Christou (Universidad de Lleida, Spain)
  • Janneke Balk y Nick Le Brun (John Innes Centre, UK)
  • Mario Piccioli (CERM, Universita degli Studi di Firenze, Italy)
  • Jose Hernandez (Midwestern University, USA)
  • Eva Kondorosi (Biological Research Centre, Szeged, Hungary)
  • Elena Caro, Manuel González Guerrero, Juan Imperial, Jaime Huerta-Cepas, Luis Fernández Pacios (Centro de Biotecnología y Genómica de Plantas, Madrid, Spain)