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

Abstract

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. '

Magnetic Properties of [FeFe]‐Hydrogenases: A Theoretical Investigation Based on Extended QM and QM/MM Models of the H‐Cluster and Its Surroundings

“…[14,[17][18][19] As noted in previous studies, reproduction of the experimental EPR g factors and hyperfine couplings of hydrogenases is a challenging task; [14,[17][18][19][20] in particular, previous theoretical studies based on simple models of the [2Fe] H subsite gave only partially satisfactory results for the H ox and H ox -CO states. [14,[17][18][19] This might be due to one or more of the following reasons: (i) models of the isolated [2Fe] H subcluster completely neglect the electronic effects of the [4Fe-4S] H subcluster, which are thought to play a relevant role in the [FeFe]-hydrogenase chemistry; [15,21,22] (ii) a poor reproduction of the environment of the H-cluster might prevent the fine reproduction of the geometrical and electronic features of the latter, thus affecting the quality of the magnetic properties calculations; [18,21] (iii) from a methodological point of view, the accuracy of electron densities obtained by DFT methods is also a matter of concern for the calculation of magnetic properties; (iv) finally, the possible presence of labile ligands such as metal-bound water molecules might deeply affect the H-cluster electron density, a point that has not been thoroughly investigated. The above issues have been tackled in this paper, by performing magnetic properties computation at different levels of theory and by including in the models the effects of the [4Fe-4S] H subsite and of the surrounding protein matrix.…”

Stepwise isotope editing of [FeFe]-hydrogenases exposes cofactor dynamics

“…However, configurations with (semi) bridging or equatorial H-species were considered as well (12,14,36) and may result from structural flexibility of the H-cluster (36). Such structural dynamics may facilitate apical or equatorial ligand binding at the distal iron ion and may also be relevant for O 2 inactivation of the enzymes via reactive oxygen species formation (24,29,30,60,61). Our protocol for selective preparation of H ox with eight distinct isotopic labeling patterns introduces spectroscopic probes at individual positions at the cofactor.…”

“…Further information on functional intermediates is required (11-16) and expected to emerge from spectroscopic studies on H-cluster constructs carrying siteselective isotopic reporter groups (17)(18)(19)(20) Formation of H ox -CO does not affect the formal redox state of the H-cluster, but leads to increased spin delocalization over the diiron site (28). CO binding inhibits H 2 turnover and protects the enzyme against O 2 and light-induced degradation (24,29,30).The vibrational modes of the CO and CN − ligands at the diiron site are well accessible by infrared (IR) spectroscopy because they are separated from protein backbone and liquid water bands.Infrared spectroscopy therefore has pioneered elucidation of the molecular structure of the H-cluster and identification of several redox states (24, 31). In particular, the CO stretching frequencies are highly sensitive to structural isomerism, redox transitions, ligand binding, and isotope exchange (11,12,15,18,24,31,32).…”

“…Formation of H ox -CO does not affect the formal redox state of the H-cluster, but leads to increased spin delocalization over the diiron site (28). CO binding inhibits H 2 turnover and protects the enzyme against O 2 and light-induced degradation (24,29,30).…”

“…Further information on functional intermediates is required (11-16) and expected to emerge from spectroscopic studies on H-cluster constructs carrying siteselective isotopic reporter groups (17)(18)(19)(20) Formation of H ox -CO does not affect the formal redox state of the H-cluster, but leads to increased spin delocalization over the diiron site (28). CO binding inhibits H 2 turnover and protects the enzyme against O 2 and light-induced degradation (24,29,30).…”