Electron transfer complex between nitrous oxide reductase and cytochrome c(552) from Pseudomonas nautica: Kinetic, nuclear magnetic resonance, and docking studies,
Dell'Acqua, Simone, Pauleta Sofia R., Monzani Enrico, Pereira Alice S., Casella Luigi, Moura Jose J. G., and Moura Isabel
, Biochemistry, Volume {47}, Number {41}, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, p.{10852-10862}, (2008)
AbstractThe multicopper enzyme nitrous oxide reductase (N2OR) catalyzes the final step of denitrification, the two-electron reduction of N2O to N-2. This enzyme is a functional homodimer containing two different multicopper sites: CuA and CuZ. CuA is a binuclear copper site that transfers electrons to the tetranuclear copper sulfide CuZ, the catalytic site. In this study, Pseudomonas nautica cytochrome C-552 was identified as the physiological electron donor. The kinetic data show differences when physiological and artificial electron donors are compared [cytochrome vs methylviologen (MV)]. In the presence of cytochrome c(552), the reaction rate is dependent on the ET reaction and independent of the N2O concentration. With MV, electron donation is faster than substrate reduction. From the study of cytochrome c(552) concentration dependence, we estimate the following kinetic parameters: K-mc512 = 50.2 +/- 9.0 mu M and V-maxc551 1.8 +/- 10.6 units/mg. The N2O concentration dependence indicates a K-mN2O of 14.0 +/- 2.9 mu M using MV as the electron donor. The pH effect on the kinetic parameters is different when MV or cytochrome c(552) is used as the electron donor (pK(a) = 6.6 or 8.3, respectively). The kinetic study also revealed the hydrophobic nature of the interaction, and direct electron transfer studies showed that CuA is the center that receives electrons from the physiological electron donor. The formation of the electron transfer complex was observed by H-1 NMR protein-protein titrations and was modeled with a molecular docking program (BiGGER). The proposed docked complexes corroborated the ET studies giving a large number of solutions in which cytochrome c(552) is placed near a hydrophobic patch located around the CuA center.
A new type of metal-binding site in cobalt- and zinc-containing adenylate kinases isolated from sulfate-reducers Desulfovibrio gigas and Desulfovibrio desulfuricans ATCC 27774,
Gavel, Olga Yu, Bursakov Sergey A., Di Rocco Giulia, Trincao Jose, Pickering Ingrid J., George Graham N., Calvete Juan J., Shnyrov Valery L., Brondino Carlos D., Pereira Alice S., Lampreia Jorge, Tavares Pedro, Moura Jose J. G., and Moura Isabel
, Journal Of Inorganic Biochemistry, Volume {102}, Number {5-6}, p.{1380-1395}, (2008)
AbstractAdenylate kinase (AK) mediates the reversible transfer of phosphate groups between the adenylate nucleotides and contributes to the maintenance of their constant cellular level, necessary for energy metabolism and nucleic acid synthesis. The AK were purified from crude extracts of two sulfate-reducing bacteria (SRB), Desulfovibrio (D.) gigas NCIB 9332 and Desulfovibrio desulfuricans ATCC 27774, and biochemically and spectroscopically characterised in the native and fully cobalt- or zinc-substituted forms. These are the first reported adenylate kinases that bind either zinc or cobalt and are related to the subgroup of metal-containing AK found, in most cases, in Gram-positive bacteria. The electronic absorption spectrum is consistent with tetrahedral coordinated cobalt, predominantly via sulfur ligands, and is supported by EPR. The involvement of three cysteines in cobalt or zinc coordination was confirmed by chemical methods. Extended X-ray absorption fine structure (EXAFS) indicate that cobalt or zinc are bound by three cysteine residues and one histidine in the metal-binding site of the ``LID'' domain. The sequence (129)Cys-X(5)-His-X(15)-Cys-X(2)-Cys of the AK from D. gigas is involved in metal coordination and represents a new type of binding motif that differs from other known zinc-binding sites of AK. Cobalt and zinc play a structural role in stabilizing the LID domain. (C) 2008 Elsevier Inc. All rights reserved.