The NADPH-oxidase is essential for the innate immune defence and is present in professional phagocyte cells. The activation of this complex is tightly regulated and involves phosphorylation events correlated to specific protein-protein interactions and chemical modifications. The phagocyte NADPH-oxidase has become the prototype of a family of electron transport system (NADPH-oxidase family) recently discovered in various tissues. They all share the capacity to produce superoxide radical from the reduction of molecular oxygen. The active enzyme is the result of the translocation of four cytosolic proteins to the membrane component, the so-called Flavocytochrome b558 (Cytb558). The Cytb558 is the catalytic core of the NADPH-oxidase that generates superoxide anion from oxygen by using reducing equivalent provided by NADPH via FAD and two hemes. A dysfunction of NADPH-oxidase leads to severe immune disease and to other important human pathologies. Our aim is to understand the functioning of this dynamic membrane-bound electron-transferring enzyme by studying its functional and structural properties in a cell-free system (in vitro). Several important steps in the NADPH-oxidase functioning have been identified. However, at the molecular level, many questions remain. The limiting factor for functional and structural studies is the lack of sufficient amounts of the membrane flavoprotein in stable, pure and homogeneous form. We recently overcame this bottleneck by producing the recombinant Cytb558 in a yeast expression system. Associated with the recombinant cytosolic proteins, the Cytb558 forms a totally recombinant cell-free system, free of cell signaling constraints and in which the environment can be easily controlled. Our aim is to take advantage of this new tool to elucidate, at the molecular level, the mechanisms underlying the reactive oxygen species production (involving protein-protein interaction, fatty acid activation, electron transfer reaction,…). Using a wide range of expertises (molecular biology, biochemistry, fluorescence and absorption spectroscopy, radiation biology and structural biology), we will gain new insight into the key structural and functional features of the protein components that control the activation and inhibition of the enzyme assembly processes and the coordination of the different redox partners. The methods like the stopped flow technique and pulsed radiolysis that we intend to use, have, to our knowledge, never been applied to analyze the NADPH-oxidase functioning. These are methods of choice to determine the reaction intermediates leading to the superoxide generation within the Cytb558 and to decipher the mechanism underlying the activation processes of the catalytic subunit. They are likely to yield unprecedented insight into the NADPH-oxidase biological functions. A fundamental aspect in this project is that the large scale production of the catalytic Cytb558 will enable us to make progress towards the determination of the three-dimensional structure of this membrane protein. This challenging project will combine a comprehensive set of biophysical, biochemical and structural approaches for the study of the NADPH-oxidase. These approaches will provide a better understanding of the molecular mechanisms underlying in the cellular process and will facilitate attempts at rational drug design where membrane proteins are often key target molecules.