ORIGINAL ARTICLE Docking and electron transfer studies between rubredoxin and rubredoxin:oxygen oxidoreductase Received: 17 July 2002 / Accepted: 9 December 2002 / Published online: 15 February 2003 Ó SBIC 2003 Abstract The interaction and electron transfer (ET) be- tween rubredoxin (Rd) and rubredoxin:oxygen oxido- reductase (ROO) from Desulfovibrio gigas is studied by molecular modelling techniques. Experimental kinetic assays using recombinant proteins show that the Rd reoxidation by ROO displays a bell-shaped dependence on ionic strength, suggesting a non-trivial electrostatic dependence of the interaction between these two pro- teins. Rigid docking studies reveal a prevalence for Rd to interact, in a very specific way, with the surface of the ROO dimer near its FMN cofactors. The optimization of the lowest energy complexes, using molecular dynamics simulation, shows a very tight interaction between the surface of the two proteins, with a high probability for Rd residues (but not the iron centre directly) to be in direct contact with the FMN cofactors of ROO. Both electrostatics and van der Waals inter- actions contribute to the final energy of the complex. In these complexes, the major contributions for complex formation are polar interactions between acidic residues of Rd and basic residues of ROO, plus substantial non- polar interactions between different groups. Important residues for this process are identified. ET estimates (using the Pathways model), in the optimized lowest energy complexes, suggest that these configurations are efficient for transferring electrons. The experimental bell-shaped dependence of kinetics on ionic strength is analysed in view of the molecular modelling results, and hypotheses for the molecular basis of this phenomenon are discussed. Keywords Protein-protein interactions Æ Molecular dynamics refinement Æ Electron transfer Æ Oxygen and NO metabolism Æ Ionic strength dependence Introduction Electron transfer (ET) between redox proteins is a bio- logical phenomenon present in multiple pathways such as photosynthesis, respiration or even biological detox- ification. ET reactions have been extensively studied and different theories about how this process occurs have been proposed. In many cases, the formation of an ef- ficient interacting complex is needed for ET to occur between two or more proteins. However, it is still under discussion whether the ET process is performed by a single productive complex (as proposed for yeast cyto- chrome c/cytochrome bc 1 complex [1]) or rather by multiple efficient conformations [2, 3, 4, 5, 6, 7]. Inde- pendently of these questions, a good characterization of the interacting surface of the complexes is essential for the understanding of ET processes between redox proteins. In the present study, we focus on the interaction be- tween Desulfovibrio gigas (D. gigas) rubredoxin:oxygen oxidoreductase (ROO) [8, 9] and its redox partner ru- bredoxin (Rd) [10, 11]. D. gigas is a sulfate-reducing bacterium, initially described as a strict anaerobe [12] (referenced in [13]). However, later studies reported the ability for this organism not only to survive in aerobic conditions, but also to reduce oxygen to water with ATP production [13, 14, 15]. These observations led to the J Biol Inorg Chem (2003) 8: 475–488 DOI 10.1007/s00775-002-0440-5 Bruno L. Victor Æ Joa˜o B. Vicente Æ Rute Rodrigues Solange Oliveira Æ Claudina Rodrigues-Pousada Carlos Fraza˜o Æ Cla´udio M. Gomes Miguel Teixeira Æ Cla´udio M. Soares B.L. Victor Æ J.B. Vicente Æ R. Rodrigues Æ S. Oliveira C. Rodrigues-Pousada Æ C. Fraza˜o Æ C.M. Gomes M. Teixeira Æ C.M. Soares (&) Instituto de Tecnologia Quı´mica e Biolo´gica, Universidade Nova de Lisboa, Apt 127, Av. Repu´blica, 2781-901, Oeiras, Portugal E-mail: claudio@itqb.unl.pt Tel.: +351-21-4469610 Fax: +351-21-4411277 S. Oliveira Departamento de Biologia, Universidade de E ´ vora, 7000, E ´ vora, Portugal C.M. Gomes Departamento de Quı´mica, Faculdade de Cieˆncias e Tecnologia, Universidade Nova de Lisboa, 2825-114 Caparica, Portugal