Spin-spin interactions between the Ni site and the [4Fe-4S] centers as a probe of light-induced structural changes in...

In typical NiFe hydrogenases like that from Desulfovibrio gigas, the active state of the enzyme which is obtained by incubation under hydrogen gas gives a characteristic Ni-C electron paramagnetic resonance (EPR) signal at g = 2.19, 2.14, and 2.01. The Ni-C species is light-sensitive, being converted upon illumination at temperatures below 100 K in a mixture of different Ni-L species, the most important giving an EPR signal at g = 2.30, 2.12, and 2.05. This photoprocess is considered to correspond to the dissociation of a hydrogen species initially coordinated to the Ni ion in the Ni-C state. When the [4Fe-4S] centers of the enzyme are reduced, the proximal [4Fe-4S]1+ cluster interacts magnetically with the Ni center, which leads to complex split Ni-C or split Ni-L EPR spectra only detectable below 10 K. In order to probe the structural changes induced in the Ni center environment by the photoprocess, these spin-spin interactions were analyzed in D. gigas hydrogenase by simulating the split Ni-L spectra recorded at different microwave frequencies. We shown that, upon illumination, the relative arrangement of the Ni and [4Fe-4S] centers is not modified but that the exchange interaction between them is completely canceled. Moreover, the rotations undergone by the Ni center magnetic axes in the photoconversion were determined. Taken together, our results support a Ni-C structure in which the hydrogen species is not in the first coordination sphere of the Ni ion but is more likely bound to a sulfur atom of a terminal cysteine ligand of the Ni center.