Spectroscopic characterization of the soluble electron transfer domains of T. Thermophilus caa3 COX

MORGADA, MARCOS N.; VILA, ALEJANDRO J.

Congreso; ICBIC18 ? International Conference on Biological Inorganic Chemistry; 2017

The dinuclear copper center CuA is the electron entry point of cytochrome c oxidase (COX). CuA funnels the electrons from reduced cytochrome c to the subunit I of COX where O2 is then reduced to water molecules. These electron transfer (ET) reactions are highly efficient and this efficiency is given by the characteristic coordination chemistry of this center.Paramagnetic spectroscopies of the CuA center in its oxidized state (Cu+1.5-Cu+1.5) revealed the presence of two electronic levels, with different orbital symmetries, σu* and πu. EPR spectroscopy studies of ba3 COX II from Thermus thermophilus (ba3-COX II) was compatible with a ΔEσ/π of ca. 5000cm-1, thus the σu* has been considered as the only redox active. NMR spectroscopy later revealed the presence of signals with Non-Curie temperature dependence behaviour, indicating that these levels are very close in energy, and both can be populated at room temperature.1,2Further studies made with mutants of the first and second coordination sphere of the CuA site in ba3-COX II, showed that the energy gap between these two electronic levels can be fine-tuned and mutations that cause an increase in the population of the πu state are not detrimental in the ET process, demonstrating that πu state is also redox active in the ET reactions.3,4 Although, it is not clear if the presence of this electronic state facilitates the ET process with the natural redox partner cytochrome c552, since there are redox-active non-canonical interactions that hamper a proper analysis.To overcome the disadvantages of the ba3-CuA model we propose the use of the soluble domain of the caa3 COX II from Thermus thermophilus (caa3-CuA) which has two domains connected with a short protein sequence: a cupredoxin CuA harbouring domain and a cytochrome c1 domain. This protein has the two domains properly oriented and would empower the analysis of the ET process between these domains.5Here we show a complete spectroscopic characterization of the caa3-CuA full protein and its individual domains indicating that we are in a presence of a proper model to understand the involvement of the πu electron state in the ET process in COX.Acknowledgments: ANPCyT, CONICETReferences[1] Gorelsky, S.I., et al. J. Am. Chem. Soc., 128, 16452 (2006)[2] Abriata, L.A., et al. J. Am. Chem. Soc., 131, 1939 (2009)[3] Abriata, L.A., et al. Proc. Natl. Acad. Sci. USA, 43, 17348 (2012)[4] Morgada, M.N., et al. Angew. Chem. Int. Ed. Engl., 54, 9555 (2014)[5] Lyons. J.A., et al. Nature, 487, 514 (2012)