title: Mitochondrial nitric oxide production supported by reverse electron transfer.
abstract: Heart phosphorylating electron transfer particles (ETPH) produced NO at 1.2 ± 0.1 nmol NO. min(-1) mg protein(-1) by the mtNOS catalyzed reaction. These particles showed a NAD(+) reductase activity of 64 ± 3 nmol min(-1) mg protein(-1) sustained by reverse electron transfer (RET) at expenses of ATP and succinate. The same particles, without NADPH and in conditions of RET produced 0.97 ± 0.07 nmol NO. min(-1) mg protein(-1). Rotenone inhibited NO production supported by RET measured in ETPH and in coupled mitochondria, but did not reduce the activity of recombinant nNOS, indicating that the inhibitory effect of rotenone on NO production is due to an electron flow inhibition and not to a direct action on mtNOS structure. NO production sustained by RET corresponds to 20% of the total amount of NO released from heart coupled mitochondria. A mitochondrial fraction enriched in complex I produced 1.7 ± 0.2 nmol NO. min(-1) mg protein(-1) and reacted with anti-75 kDa complex I subunit and anti-nNOS antibodies, suggesting that complex I and mtNOS are located contiguously. These data show that mitochondrial NO production can be supported by RET, and suggest that mtNOS is next to complex I, reaffirming the idea of a functional association between these proteins.
authors: Institute of Biochemistry and Molecular Medicine, Physical Chemistry Division, School of Pharmacy and Biochemistry, University of Buenos Aires (IBIMOL, UBA-CONICET), Junín 956, C1113AAD, Buenos Aires, Argentina. Electronic address: sbombicino@ffyb.uba.ar.
authors: Silvina S
authors: SS
authors: Bombicino
authors: Institute of Biochemistry and Molecular Medicine, Physical Chemistry Division, School of Pharmacy and Biochemistry, University of Buenos Aires (IBIMOL, UBA-CONICET), Junín 956, C1113AAD, Buenos Aires, Argentina.
authors: Darío E
authors: DE
authors: Iglesias
authors: Institute of Biochemistry and Molecular Medicine, Physical Chemistry Division, School of Pharmacy and Biochemistry, University of Buenos Aires (IBIMOL, UBA-CONICET), Junín 956, C1113AAD, Buenos Aires, Argentina.
authors: Tamara
authors: T
authors: Zaobornyj
authors: Institute of Biochemistry and Molecular Medicine, Physical Chemistry Division, School of Pharmacy and Biochemistry, University of Buenos Aires (IBIMOL, UBA-CONICET), Junín 956, C1113AAD, Buenos Aires, Argentina.
authors: Alberto
authors: A
authors: Boveris
authors: Institute of Biochemistry and Molecular Medicine, Physical Chemistry Division, School of Pharmacy and Biochemistry, University of Buenos Aires (IBIMOL, UBA-CONICET), Junín 956, C1113AAD, Buenos Aires, Argentina.
authors: Laura B
authors: LB
authors: Valdez
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D002384:Catalysis; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D004583:Electrons; D008928:Mitochondria / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism; D064430:NAD (+) and NADP (+) Dependent Alcohol Oxidoreductases / Q000378:metabolism; D009249:NADP / Q000737:chemistry; D009569:Nitric Oxide / Q000378:metabolism*; D010101:Oxygen Consumption; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D012402:Rotenone / Q000737:chemistry; D013367:Submitochondrial Particles / Q000737:chemistry; D019802:Succinic Acid / Q000737:chemistry
chemical_list: D011994:Recombinant Proteins; D012402:Rotenone; D009569:Nitric Oxide; D009249:NADP; D000255:Adenosine Triphosphate; D019802:Succinic Acid; D064430:NAD (+) and NADP (+) Dependent Alcohol Oxidoreductases
keywords: Complex I; FMN; HbO(2); Inside-out particles; Mitochondrial nitric oxide synthase; Nitric oxide; O(2)(•-); RET; Reverse electron transfer; flavin mononucleotide; mitochondrial nitric oxide synthase; mtNOS; oxyhemoglobin; proton electrochemical potential; proton motive force; reverse electron transfer; superoxide anion; Δp; ΔμH(+)