ISBN: 978-83-60043-10-3 - eurobic9

Eurobic9, 2-6 September, 2008, Wrocław, Poland
P35. Pseudomonas nautica Cytochrome c552 is the Electron Donor to Nitrous
Oxide Reductase (N2OR), a Kinetic and Docking Study
S. Dell’Acqua a , S.R. Pauleta a , A.S. Pereira a , E. Monzani b , L. Casella b , I. Moura a ,
J.J. G. Moura a
a
REQUIMTE/CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de
Lisboa, 2829-516 Caparica, Portugal
e-mail: simone.dellacqua@dq.fct.unl.pt
b
Dipartimento di Chimica Generale, Università di Pavia, Via Taramelli 12, 27100 Pavia, Italy
The multicopper enzyme nitrous oxide reductase (N2OR) catalyses the final step of denitrification, the twoelectron
reduction of N2O to N2. This enzyme is a functional homodimer containing two different multicopper
sites: CuA and CuZ, where CuA is a binuclear copper site that transfers electrons to the tetranuclear coppersulfide
CuZ, the catalytic site.
The new activity assay presented here separates the activation of N2OR from the catalytic reduction of N2O and
allowed to identify Pseudomonas nautica cytochrome c552 as the physiological electron donor to N2OR. The
kinetic data presents differences when comparing physiological with artificial electron donors (cytochrome
versus methylviologen). In the presence of cytochrome c552, the reaction rate is dependent on the ET reaction and
independent of the N2O concentration. With MV, the electron donation is faster than the substrate reduction. The
pH effect on the kinetic parameters is different when MV or cytochrome c552 are used as electron donors
(pKa=6.6 and 8.3, respectively). The kinetic study also suggested the hydrophobic nature of the interaction. The
formation of the electron-transfer complex was observed by 1 H-NMR protein-protein titrations and was
modelled with a molecular docking program (BiGGER) [1]. The proposed docked complexes corroborated with
the ET studies, giving a cluster of solutions that places cytochrome c552 nearby a hydrophobic patch located
around the CuA center [2].
Acknowledgement: S.D.is supported by a FCT-PhD grant (SFRH/BD/30414/2006)
References :
[1] P.N. Palma et al., Proteins, 39, 372 (2000).
[2] S. Dell’Acqua et al., Biochemistry, submitted.
_____________________________________________________________________
154