title: Covalent modifications of the catalytic tyrosine in octahaem cytochrome c nitrite reductase and their effect on the enzyme activity.
abstract: Octahaem cytochrome c nitrite reductase from Thioalkalivibrio nitratireducens (TvNiR), like the previously characterized pentahaem nitrite reductases (NrfAs), catalyzes the six-electron reductions of nitrite to ammonia and of sulfite to sulfide. The active site of both TvNiR and NrfAs is formed by the lysine-coordinated haem and His, Tyr and Arg residues. The distinguishing structural feature of TvNiR is the presence of a covalent bond between the CE2 atom of the catalytic Tyr303 and the S atom of Cys305, which might be responsible for the higher nitrite reductase activity of TvNiR compared with NrfAs. In the present study, a new modified form of the enzyme (TvNiRb) that contains an additional covalent bond between Tyr303 CE1 and Gln360 CG is reported. Structures of TvNiRb in complexes with phosphate (1.45 Å resolution) and sulfite (1.8 Å resolution), the structure of TvNiR in a complex with nitrite (1.83 Å resolution) and several additional structures were determined. The formation of the second covalent bond by Tyr303 leads to a decrease in both the nitrite and sulfite reductase activities of the enzyme. Tyr303 is located at the exit from the putative proton-transport channel to the active site, which is absent in NrfAs. This is an additional argument in favour of the involvement of Tyr303 as a proton donor in catalysis. The changes in the activity of cytochrome c nitrite reductases owing to the formation of Tyr-Cys and Tyr-Gln bonds may be associated with changes in the pK(a) value of the catalytic tyrosine.
authors.affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow 119991, Russian Federation. burayatina@gmail.com
authors.forename: Anton A
authors.initials: AA
authors.lastname: Trofimov
authors.forename: Konstantin M
authors.initials: KM
authors.lastname: Polyakov
authors.forename: Tamara V
authors.initials: TV
authors.lastname: Tikhonova
authors.forename: Alexey V
authors.initials: AV
authors.lastname: Tikhonov
authors.forename: Tatyana N
authors.initials: TN
authors.lastname: Safonova
authors.forename: Konstantin M
authors.initials: KM
authors.lastname: Boyko
authors.forename: Pavel V
authors.initials: PV
authors.lastname: Dorovatovskii
authors.forename: Vladimir O
authors.initials: VO
authors.lastname: Popov
mesh_terms: D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D045263:Cytochromes a1 / Q000737:chemistry* / Q000378:metabolism*; D003575:Cytochromes c1 / Q000737:chemistry* / Q000378:metabolism*; D042001:Ectothiorhodospiraceae / Q000737:chemistry / Q000201:enzymology*; D008958:Models, Molecular; D009565:Nitrate Reductases / Q000737:chemistry* / Q000378:metabolism*; D009573:Nitrites / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D011485:Protein Binding; D013447:Sulfites / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry* / Q000378:metabolism
chemical_list: D009573:Nitrites; D010710:Phosphates; D013447:Sulfites; D014443:Tyrosine; D045263:Cytochromes a1; D003575:Cytochromes c1; D009565:Nitrate Reductases; C054690:nitrate reductase (cytochrome)