The implication of the tyrosil radical in the microcin J25 molecule

CHALON MIRIAM CAROLINA; CORTEZ LEONARDO; BELLOMIO AUGUSTO; MORERO ROBERTO; CHEIN ROSSANA; FARÍAS RICARDO; VINCENT PAULA

San Miguel de Tucumán - Argentina

Congreso; Reunión Anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular.; 2009

Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular.

Our previous results showed that MccJ25 targets the bacterial membrane respiratory chain enzymes (NADH and lactate dehydrogenase activities), inhibiting cell oxygen consumption. This effect is mediated by an increase in superoxide production by the  respiratory processes. We also demonstrated that Tyr9 of MccJ25 is essential for the action on the respiratory chain. In this work, we investigated the tyrosil radical formation using Electronic Paramagnetic Resonance (EPR) and Fourier transform infrared spectroscopy (2D-FTIR). The tyrosil radical was detected by EPR when 2.4 mM MccJ25 was incubated in the presence of 2.5 mM H2O2 and 1 mM DBNBS (trapping of the tyrosil radical). Moreover, FTIR experiments showed an increment of tyrosil radical band located at 1,478 cm-1 in the spectrum. To confirm that only Tyr9 is the responsible of tyrosil radical formation, we carried out the reaction with the MccJ25 (Y9F) mutant and no tyrosil radical was detected. On other hand, MccJ25 was unable to inhibited enzymes activities when the experiment was performed with membrane from an E. coli cydAB. These results confirm the presence of a tyrosil radical in MccJ25 molecule and suggest that it could be involved in the superoxide overproduction induced by the antibiotic. Moreover, we proposed that cytochrome bd is essential for the MccJ25 effect on respiratory chain enzymes.