Reduction of O2 and NO in flavodiiron proteins - Tuning the energy landscape by second sphere ligation variations

Flavodiiron proteins (FDPs) constitute a large family of non-heme iron enzymes present in all domains of life. They play important roles as scavengers and detoxifiers by efficiently reducing both O${}_2$ and NO. The primary ligands of the diiron active site in all FDPs are highly conserved, indicating that the basic reaction mechanisms for O${}_2$ and NO reduction, respectively, are the same. However, the reduction activity varies significantly between different FDPs. By comparing FDPs from two different species, Thermotoga maritima and Desulfovibrio gigas, we investigate to what extent variations in the second sphere ligation can explain differences in reduction activities. Comparisons are also made between wildtype and two variants of Thermotoga maritima FDP. We use Density functional theory (DFT) calculations on a number of FDP active site models to study the reaction mechanisms for both O${}_2$ and NO reduction. For reduction of O${}_2$ we conclude that differences in activity cannot be explained by differences in the first or second active site coordination spheres, which is mainly due to a low barrier for OO bond cleavage after one proton-coupled reduction step. For NO reduction however, the rate-limiting barrier for N${}_2$O formation, a hyponitrite rotation, is high enough to be involved in the overall rate limitation. We show that second sphere residues, such as Tyr26 in Desulfovibrio gigas FDP, that can form hydrogen bonds to the rotating hyponitrite, decrease the barrier. Differences in NO reduction rate among different FDPs are most likely determined by the variation in such second sphere residues.