The International Journal of Biochemistry & Cell Biology 76 (2016) 12–18 Contents lists available at ScienceDirect The International Journal of Biochemistry & Cell Biology jo u r n al homep ag e: www.elsevier.com/locate/biocel Stimulation by pro-apoptotic valinomycin of cytosolic NADH/cytochrome c electron transport pathway—Effect of SH reagents Dario Domenico Lofrumento a , Gianluigi La Piana b , Valeria Palmitessa b , Daniela Isabel Abbrescia b , Nicola Elio Lofrumento b,∗ a Department of Biological and Environmental Sciences and Technologies, Section of Human Anatomy, University of Salento, Lecce, Italy b Department of Biosciences, Biotechnology and Biopharmaceutics, University of Bari, Bari, Italy a r t i c l e i n f o Article history: Received 3 February 2016 Received in revised form 14 April 2016 Accepted 25 April 2016 Available online 26 April 2016 Keywords: Apoptosis Valinomycin Cytochrome c Cytosolic NADH oxidation Mitochondrial contact sites SH inhibitors a b s t r a c t Intrinsic and extrinsic apoptosis are both characterised by the presence of cytochrome c (cyto-c) in the cytosol. We present data on the extra-mitochondrial NADH oxidation catalysed by exogenous (cytoso- lic) cyto-c, as a possible answer to the paradox of apoptosis being an energy-dependent program but characterized by the impairment of the respiratory chain. The reduction of molecular oxygen induced by the cytosolic NADH/cyto-c pathway is coupled to the generation of an electrochemical proton gradient available for ATP synthesis. Original findings show that SH reagents inhibit the NADH/cyto-c system with a conformational change mechanism. The mitochondrial integrity-test of sulfite oxidase unequivocally demonstrates that this enzyme (120 kDa) can be released outside but exogenous cyto-c (12.5 kDa) does not permeate into mitochondria. Valinomycin at 2 nM stimulates both the energy-dependent reversible mitochondrial swelling and the NADH/cyto-c oxidation pathway. The pro-apoptotic activity of valino- mycin, as well as to the dissipation of membrane potential, can be also ascribed to the increased activity of the NADH/cyto-c oxidation pathway useful as an additional source of energy for apoptosis. It can be speculated that the activation of the NADH/cyto-c system coupled to valinomycin-induced mitochondrial osmotic swelling may represent a strategy to activate apoptosis in confined solid tumours. © 2016 Published by Elsevier Ltd. 1. Introduction Permeabilization of the mitochondrial outer membrane (MOM) remains one of the first events of both the intrinsic and extrinsic pathways of apoptosis driving the release of pro-apoptotic pro- teins, including cytochrome c (cyto-c), from the mitochondrial intermembrane space (MIS) (Ow et al., 2008). The discovery of the involvement of cytosolic cyto-c remains a milestone in the elu- cidation of the programmed cell death (Liu et al., 1996). It was definitively shown that cyto-c is not present exclusively in the MIS Abbreviations: cyto-c, cytochrome c; MOM, mitochondrial outer membrane; MIS, mitochondrial intermembrane space; MIM, mitochondrial inner mem- brane; m, mitochondrial membrane potential; p, electrochemical proton gradient; porin, voltage dependent anion channel; Sox, sulfite oxidase; NEM, N- ethylmaleimide; Mrs, mersalyl. ∗ Corresponding author at: Department of Biosciences, Biotechnology and Bio- pharmaceutics, University of Bari, via Orabona 4, 70126 Bari, Italy. E-mail address: nicolaelio.lofrumento@uniba.it (N.E. Lofrumento). but mobilized by cleavage of OPA1 protein (Frezza et al., 2006) participates in fundamental processes inside the cytosol. Cumulative data are consistent with the direct oxidation of cytosolic NADH catalysed by cyto-c molecules present outside the mitochondria. This system promotes the reduction of molecular oxygen, generates an electrochemical proton gradient available for ATP synthesis and is inhibited by cyanide but not by other res- piratory chain inhibitors (Bodrova et al., 1998; Gorgoglione et al., 2007; La Piana et al., 1998, 2005; Lemeshko et al., 2003; Lofrumento et al., 1991; Marzulli et al., 1995). The main components of the system may reside at specific contact points between the two mitochondrial membranes indicated as “respiratory contact sites” (Gorgoglione et al., 2010; La Piana et al., 2005; Marzulli et al., 1999). The exogenous NADH oxidation supported by the malate- aspartate shuttle is comparable to that of the NADH/cyto-c system (Abbrescia et al., 2012). Note that the malate-aspartate shuttle is an indirect pathway since the reducing equivalents are first trans- ferred to oxaloacetate, converted into malate which is relocated to and oxidized inside the mitochondria generating NADH in the matrix to be oxidized by Complex I. Then electrons are sent via the http://dx.doi.org/10.1016/j.biocel.2016.04.014 1357-2725/© 2016 Published by Elsevier Ltd.