title: Redox properties of lysine- and methionine-coordinated hemes ensure downhill electron transfer in NrfH2A4 nitrite reductase.
abstract: The multiheme NrfHA nitrite reductase is a menaquinol:nitrite oxidoreductase that catalyzes the 6-electron reduction of nitrite to ammonia in a reaction that involves eight protons. X-ray crystallography of the enzyme from Desulfovibrio vulgaris revealed that the biological unit, NrfH2A4, houses 28 c-type heme groups, 22 of them with low spin and 6 with pentacoordinated high spin configuration. The high spin hemes, which are the electron entry and exit points of the complex, carry a highly unusual coordination for c-type hemes, lysine and methionine as proximal ligands in NrfA and NrfH, respectively. Employing redox titrations followed by X-band EPR spectroscopy and surface-enhanced resonance Raman spectroelectrochemistry, we provide the first experimental evidence for the midpoint redox potential of the NrfH menaquinol-interacting methionine-coordinated heme (-270 ± 10 mV, z = 0.96), identified by the use of the inhibitor HQNO, a structural analogue of the physiological electron donor. The redox potential of the catalytic lysine-coordinated high spin heme of NrfA is -50 ± 10 mV, z = 0.9. These values determined for the integral NrfH2A4 complex indicate that a driving force for a downhill electron transfer is ensured in this complex.
authors.affiliation: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa , Av. da Republica, 2780-157 Oeiras, Portugal.
authors.forename: Smilja
authors.initials: S
authors.lastname: Todorovic
authors.forename: Maria Luísa
authors.initials: ML
authors.lastname: Rodrigues
authors.forename: Daniela
authors.initials: D
authors.lastname: Matos
authors.forename: Inês A C
authors.initials: IA
authors.lastname: Pereira
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D045263:Cytochromes a1 / Q000737:chemistry*; D003575:Cytochromes c1 / Q000737:chemistry*; D016969:Desulfovibrio vulgaris / Q000201:enzymology*; D004583:Electrons; D006418:Heme / Q000737:chemistry*; D006912:Hydroxyquinolines / Q000737:chemistry; D008958:Models, Molecular; D009565:Nitrate Reductases / Q000737:chemistry*; D010084:Oxidation-Reduction; D011487:Protein Conformation; D012996:Solutions; D012997:Solvents / Q000737:chemistry; D013057:Spectrum Analysis; D013059:Spectrum Analysis, Raman; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D006912:Hydroxyquinolines; D012996:Solutions; D012997:Solvents; C001333:2-(n-heptyl)-4-hydroxyquinoline N-oxide; D006418:Heme; D045263:Cytochromes a1; D003575:Cytochromes c1; D009565:Nitrate Reductases; C054690:nitrate reductase (cytochrome)