H+ ions and electrons released from NADH and FADH2,on to the inner mitochondrial membrane. electrons enter electron transport chain, and energy released from transfer of electrons is used to pump H+ ions into the inter-membrane space. as the inner mitochondrial membrane is impermeable to protons, H+ ions cannot pass back through the inner mitochondrial membrane, into the matrix, therefore there is a higher concentration of H+ ions in the inter-membrane space, thus setting up and maintaining a proton gradient across the inner mitochondrial membrane.