title: Structure and kinetic properties of Paracoccus pantotrophus cytochrome cd1 nitrite reductase with the d1 heme active site ligand tyrosine 25 replaced by serine.
abstract: The 1.4-A crystal structure of the oxidized state of a Y25S variant of cytochrome cd(1) nitrite reductase from Paracoccus pantotrophus is described. It shows that loss of Tyr(25), a ligand via its hydroxy group to the iron of the d(1) heme in the oxidized (as prepared) wild-type enzyme, does not result in a switch at the c heme of the unusual bishistidinyl coordination to the histidine/methionine coordination seen in other conformations of the enzyme. The Ser(25) side chain is seen in two positions in the d(1) heme pocket with relative occupancies of approximately 7:3, but in neither case is the hydroxy group bound to the iron atom; instead, a sulfate ion from the crystallization solution is bound between the Ser(25) side chain and the heme iron. Unlike the wild-type enzyme, the Y25S mutant is active as a reductase toward nitrite, oxygen, and hydroxylamine without a reductive activation step. It is concluded that Tyr(25) is not essential for catalysis of reduction of any substrate, but that the requirement for activation by reduction of the wild-type enzyme is related to a requirement to drive the dissociation of this residue from the active site. The Y25S protein retains the d(1) heme less well than the wild-type protein, suggesting that the tyrosine residue has a role in stabilizing the binding of this cofactor.
authors.affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.
authors.forename: Euan H J
authors.initials: EH
authors.lastname: Gordon
authors.forename: Tove
authors.initials: T
authors.lastname: Sjögren
authors.forename: Malin
authors.initials: M
authors.lastname: Löfqvist
authors.forename: Carsten D
authors.initials: CD
authors.lastname: Richter
authors.forename: James W A
authors.initials: JW
authors.lastname: Allen
authors.forename: Christopher W
authors.initials: CW
authors.lastname: Higham
authors.forename: Janos
authors.initials: J
authors.lastname: Hajdu
authors.forename: Vilmos
authors.initials: V
authors.lastname: Fülöp
authors.forename: Stuart J
authors.initials: SJ
authors.lastname: Ferguson
mesh_terms: D001665:Binding Sites / Q000502:physiology; D003580:Cytochromes; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006418:Heme / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D016296:Mutagenesis / Q000502:physiology; D009572:Nitrite Reductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010230:Paracoccus / Q000201:enzymology*; D017434:Protein Structure, Tertiary; D012694:Serine / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D014443:Tyrosine / Q000378:metabolism
chemical_list: D003580:Cytochromes; D008024:Ligands; D014443:Tyrosine; D006418:Heme; D012694:Serine; D009572:Nitrite Reductases; C028123:nitrite reductase (NO-forming); D003576:Electron Transport Complex IV