Life and the Chemistry of Oxygen

This proposal addresses the question of how oxygen is activated by the living cell. Not only is oxygen indispensable for respiration it is also used in many anabolic and catabolic reactions. The problem with oxygen is that it has a high activation barrier. The cell must find ways to overcome this and it does so by employing enzymes that lower this barrier. An important class of these enzymes consists of the so-called multi-metal enzymes that catalyze the conversion of oxygen to water. In this proposal we focus on the multi-copper enzymes in which the oxygen is captured by a tri-nuclear Cu site and subsequently reduced by four electrons to produce water. The details of this reaction are unclear. A better understanding of what happens during the oxygen conversion is not only relevant for a better understanding of the (bio)chemistry of oxygen, it is also important from a technical point of view. A better understanding of oxygen chemistry may be relevant for applications were oxygen plays a critical role like the design of fuel cells. A crucial question is if the metals in the enzyme are sufficient to initiate and complete the oxygen reduction or whether assistance from a enzyme-derived radical is required. Circumstantial evidence suggests that such a radical may be necessary, but definitive proof despite efforts by many groups in this direction, is lacking until now. Recently we have identified a copper enzyme that turns out to be a representative of a new class of multi-copper oxidases, the so-called small laccases (SLACs). SLAC turns out to be an excellent candidate to investigate the mechanism of the oxygen-to-water conversion. It appears to be the first example where the spectroscopic signature of a radical intermediate has been observed beyond doubt in the reaction of the reduced enzyme with oxygen. We think that we are in an excellent position to shed fundamental new light on the reductive chemistry of oxygen. The key features that mark our position are " we have a unique model system at hand to study this reaction in the form of the SLAC enzyme from Streptomyces coelicolor. The production and purification of the enzyme have been explored in detail by us; we have developed a good-working cloning system to prepare site-directed variants of SLAC; the 3-dimensional structure of the enzyme has been reported very recently. The latter forms an excellent basis to correlate spectroscopic and mechanistic findings with structural details; " the occurrence of a radical intermediate has been established by us beyond doubt; " the methods of choice to study paramagnetic proteins are EPR and NMR. We have the advantage that we avail of almost unique facilities for high frequency (pulsed and CW) EPR and ENDOR studies of enzymes. When applied to SLAC they will give invaluable information about spin density distribution in the active site and the magnetic properties of the metal centres as the reaction of oxygen in the site proceeds; " NMR of paramagnetic proteins is a specialty of the Leiden group. We have found that paramagnetically shifted and well-resolved signals occur in the NMR-spectrum of SLAC. In principle they contain detailed information not only about the spin density distribution in the active site but also about the structure of the metal coordination. When combining the information to be gleaned form the magnetic resonance experiments on carefully designed and produced site-directed SLAC variants with stopped flow studies, crucial new information can be obtained about the mode of action of oxygen activating enzymes and the reductive chemistry of oxygen.