Deciphering mechanisms of respiratory chain complexes by IR labelling and surface enhanced approaches

The aerobic respiratory chain of E. coli consists of several primary dehydrogenases and terminal reductases. Complex I is the largest enzyme in the respiratory chain that catalyzes the transfer of two electrons from NADH to quinone coupled with the translocation of four protons across the membrane. It has an L-shape structure consisting of a peripheral and a membrane arm. The coupling of both processes is still under debate. A nitrile label was inserted in the proximity of one of the putative proton pathways in NuoM of complex I. Individual positions were genetically changed to cysteines and labelled with cyanides. Nitrile labels are attractive IR labels as they can provide valuable information about the local environment of the probe in a defined protein environment. Conformational changes in these positions were visualized with SEIRAS upon addition of substrates. Cytochrome bd-I oxidase belongs to terminal reductases of respiratory chains. Conserved glutamic acid residues in proximity of hemes are important for functions of bd-I oxidase. These findings are discussed in relation to the physiological role of the enzyme.