Spectroscopic investigations of a semi-synthetic [FeFe] hydrogenase with propane di-selenol as bridging ligand in the binuclear subsite: comparison to the wild type and propane di-thiol variants

International audience[FeFe] Hydrogenases catalyze the reversible conversion of H$_2$ into electrons and protons. Their catalytic site, the H-cluster, contains a generic [4Fe–4S]$_H$ cluster coupled to a [2Fe]$_H$ subsite [Fe$_2$(ADT)(CO)$_3$(CN)$_2$]$^{2−}$, ADT = $\mu$(SCH$_2$)$_2$NH. Heterologously expressed [FeFe] hydrogenases (apo-hydrogenase) lack the [2Fe]$_H$ unit, but this can be incorporated through artificial maturation with a synthetic precursor [Fe$_2$(ADT)(CO)$_4$(CN)$_2$]$^{2−}$. Maturation with a [2Fe] complex in which the essential ADT amine moiety has been replaced by CH$_2$ (PDT = propane-dithiolate) results in a low activity enzyme with structural and spectroscopic properties similar to those of the native enzyme, but with simplified redox behavior. Here, we study the effect of sulfur-to-selenium (S-to-Se) substitution in the bridging PDT ligand incorporated in the [FeFe] hydrogenase HydA1 from $Chlamydomonas\ reinhardtii$ using magnetic resonance (EPR, NMR), FTIR and spectroelectrochemistry. The resulting HydA1-PDSe enzyme shows the same redox behavior as the parent HydA1-PDT. In addition, a state is observed in which extraneous CO is bound to the open coordination site of the [2Fe]$_H$ unit. This state was previously observed only in the native enzyme HydA1-ADT and not in HydA1-PDT. The spectroscopic features and redox behavior of HydA1-PDSe, resulting from maturation with [Fe$_2$(PDSe)(CO)$_4$(CN)$_2$]$^{2−}$, are discussed in terms of spin and charge density shifts and provide interesting insight into the electronic structure of the H-cluster. We also studied the effect of S-to-Se substitution in the [4Fe–4S] subcluster. The reduced form of HydA1 containing only the [4Fe–4Se]$_H$ cluster shows a characteristic S = 7/2 spin state which converts back into the S = 1/2 spin state upon maturation with a [2Fe]–PDT/ADT complex