Biochemical characterization of mitochondrial complex III inhibition by nitric oxide.

IGLESIAS DE; BOMBICINO SS; BOVERIS A; VALDEZ LB

Mendoza

Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB).; 2012

Sociedad Argentina de Investigacion en Bioquimica y Biologia Molecular

Little is known about how nitric oxide (NO) interacts with the NO-reactive component of mitochondrial complex III. The aim of this work was to study the inhibitory effect of NO on electron transfer between cyt. b and cyt. c using beef heart inside-out particles (ETPH-Mg2+). Succinate-cytochrome c reductase activity (222±4 nmol/min.mg protein) was inhibited (51%) by 500 mM GSNO; this activity was also reduced (36%) when ETPH-Mg2+ were incubated in the presence of L-arg and mtNOS cofactors, suggesting that this effect is caused by mtNOS-produced NO. In mitochondrial membranes, ~240 nM NO reduced cyt. b562 by 70%. The effective [NO] was assessed using a NO-sensitive electrode: 500 mM GSNO releases 240 nM NO to the reaction medium when the assay is achieved during 2 min. NO produced an increase in O2- and H2O2 production rates with a maximal effect at 500 mM GSNO (1.3±0.1 nmol O2-/min.mg protein; 0.64±0.05 nmol H2O2/min.mg protein). The O2-/H2O2 ratio was 2.0 in accordance to the stoichiometry of the O2- dismutation reaction. ETPH-Mg2+ incubated in the presence of succinate showed an EPR signal at g~1.99, compatible with a stable semiquinone (UQH·), which was increased by GSNO. The interaction of NO with complex III leads to electron transfer inhibition, in an [O2] independent manner, with a [UQH·]ss enhancement, which in turn, generates an increase in O2- and H2O2 production rates.