title: Detection of the His-heme Fe2+-NO species in the reduction of NO to N2O by ba3-oxidase from thermus thermophilus.
abstract: Reaction pathways in the enzymatic formation and cleavage of the N-N and N-O bonds, respectively, are difficult to verify without the structure of the intermediates, but we now have such information on the heme a(3)(2+)-NO species formed in the reaction of ba(3)-oxidase with NO from resonance Raman spectroscopy. We have identified the His-heme a(3)(2+)-NO/Cu(B)(1+) species by its characteristic Fe-NO and N-O stretching frequencies at 539 and 1620 cm(-)(1), respectively. The Fe-NO and N-O frequencies in ba(3)-oxidase are 21 and 7 cm(-)(1) lower and higher, respectively, than those observed in Mb-NO. From these results and earlier Raman and FTIR measurements, we demonstrate that the protein environment of the proximal His384 that is part of the Q-proton pathway controls the strength of the Fe-His384 bond upon ligand (CO vs NO) binding. We also show by time-resolved FTIR spectroscopy that Cu(B)(1+) has a much lower affinity for NO than for CO. We suggest that the reduction of NO to N(2)O by ba(3)-oxidase proceeds by the fast binding of the first NO molecule to heme a(3) with high-affinity, and the second NO molecule binds to Cu(B) with low-affinity, producing the temporal co-presence of two NO molecules in the heme-copper center. The low-affinity of Cu(B) for NO binding also explains the NO reductase activity of the ba(3)-oxidase as opposed to other heme-copper oxidases. With the identification of the His-heme a(3)(2+)-NO/Cu(B)(1+) species, the structure of the binuclear heme a(3)-Cu(B)(1+) center in the initial step of the NO reduction mechanism is known.
authors.affiliation: Department of Chemistry, University of Crete, 71409 Heraklion, Crete, Greece.
authors.forename: Eftychia
authors.initials: E
authors.lastname: Pinakoulaki
authors.forename: Takehiro
authors.initials: T
authors.lastname: Ohta
authors.forename: Tewfik
authors.initials: T
authors.lastname: Soulimane
authors.forename: Teizo
authors.initials: T
authors.lastname: Kitagawa
authors.forename: Constantinos
authors.initials: C
authors.lastname: Varotsis
mesh_terms: D001665:Binding Sites; D003300:Copper / Q000737:chemistry; D003573:Cytochrome b Group / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000378:metabolism*; D005296:Ferrous Compounds / Q000737:chemistry; D006418:Heme / Q000031:analogs & derivatives*; D006639:Histidine / Q000737:chemistry*; D008024:Ligands; D050901:Nitrate Reductase / Q000378:metabolism; D009589:Nitrogen Oxides / Q000737:chemistry*; D009609:Nitrous Oxide / Q000737:chemistry*; D010084:Oxidation-Reduction; D011522:Protons; D017550:Spectroscopy, Fourier Transform Infrared; D013059:Spectrum Analysis, Raman; D016963:Thermus thermophilus / Q000201:enzymology
chemical_list: D003573:Cytochrome b Group; D005296:Ferrous Compounds; D008024:Ligands; D009589:Nitrogen Oxides; D011522:Protons; D006418:Heme; D006639:Histidine; D003300:Copper; C058557:cytochrome ba3; D050901:Nitrate Reductase; D003576:Electron Transport Complex IV; D009609:Nitrous Oxide