A MccJ25 tyrosil radical is involved in the respiratory chain inhibition by the antimicrobial peptide

CHALON MIRIAM CAROLINA; WILKE NATALIA; NIKLISON CHIROU M.V; CORTEZ LEONARDO; BELLOMIO AUGUSTO; MORERO ROBERTO; CHEIN ROSSANA; FARÍAS RICARDO; VINCENT PAULA

Los cocos, Cordoba, Argentina

Congreso; Reunión Anual de la Sociedad Argentina de Biofísica; 2009

Sociedad Argentina de Biofísica

MccJ25 is an antimicrobial peptide that targets both, the DNA transcription process and the bacterial membrane respiratory chain, inhibiting cell oxygen consumption. This inhibition is mediated by an increase in superoxide production by the respiratory processes. It was demonstrated that Tyr9 of MccJ25 plays an essential role in the action of the peptide on the respiratory chain. A redox-active tyrosine residue is important in the catalysis mechanism of several enzymes, including ribonucleotide reductase, prostaglandin H synthase, and Photosystem II [1]. Here, we investigated the tyrosil radical formation in MccJ25 using Electronic Paramagnetic Resonance (EPR) and Fourier transform infrared spectroscopy (2D-FTIR). The tyrosil radical was detected by EPR when MccJ25 was incubated in the presence of H2O2 and DBNBS (a trapping tyrosil radical agent). In addition, FTIR experiments showed an increment of tyrosil radical band located at 1,478 cm-1 in the spectrum. Moreover, the redox potential of MccJ25 was determinated by cyclic voltammetry techniques and an irreversible oxidation at about 970 mV vs calomel electrode was detected, a value corresponding to tyrosine oxidation. On other hand, MccJ25 was unable to inhibited respiratory chain enzymes activities when the experiments were performed with membranes obtained from E. coli deficient in cytochrome bd and quinones production pathways. All these results confirmed the presence of a tyrosil radical in the MccJ25 molecule and suggest that it could be formed by oxidation in the respiratory chain. Besides, we propose that cytochrome bd and quinores are essential for the MccJ25 effect on respiratory chain. [1] Vassiliev, I.  R., Offenbacher, A. R., Barry, B. A.,J. Phys. Chem. B., 2005,109:23077-23085.