Spin distribution of the H-cluster in the H_ox-CO state of the [FeFe] hydrogenase from Desulfovibrio desulfuricans: HYSCORE and ENDOR study of ¹⁴N and ¹³C nuclear interactions

Hydrogenases are enzymes which catalyze the reversible cleavage of molecular hydrogen into protons and electrons. In [FeFe] hydrogenases the active center is a 6Fe6S cluster, referred to as the "H-cluster." It consists of the redox-active binuclear subcluster ([2Fe]_H) coordinated by CN^- and CO ligands and the cubane-like [4Fe-4S] _H subcluster which is connected to the protein via Cys ligands. One of these Cys ligands bridges to the [2Fe]_H subcluster. The CO-inhibited form of [FeFe] hydrogenase isolated from Desulfovibrio desulfuricans was studied using advanced EPR methods. In the H_ox-CO state the open coordination site at the [2Fe]_H subcluster is blocked by extrinsic CO, giving rise to an EPR-active S = 1/2 species. The CO inhibited state was prepared with ¹³CO and illuminated under white light at 273 K. In this case scrambling of the CO ligands occurs. Three ¹³C hyperfine couplings of 17.1, 7.4, and 3.8 MHz (isotropic part) were observed and assigned to ¹³CO at the extrinsic, the bridging, and the terminal CO-ligand positions of the distal iron, respectively. No ¹³CO exchange of the CO ligand to the proximal iron was observed. The hyperfine interactions detected indicate a rather large distribution of the spin density over the terminal and bridging CO ligands attached to the distal iron. Furthermore, ¹⁴N nuclear spin interactions were measured. On the basis of the observed ¹⁴N hyperfine couplings, which result from the CN^- ligands of the [2Fe]_H subcluster, it has been concluded that there is very little unpaired spin density on the cyanides of the binuclear subcluster.