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

Abstract

[FeFe] Hydrogenases catalyze the reversible conversion of H2 into electrons and protons. Their catalytic site, the H-cluster, contains a generic [4Fe–4S]H cluster coupled to a [2Fe]H subsite [Fe2(ADT)(CO)3(CN)2]2−, ADT = µ(SCH2)2NH. Heterologously expressed [FeFe] hydrogenases (apo-hydrogenase) lack the [2Fe]H unit, but this can be incorporated through artificial maturation with a synthetic precursor [Fe2(ADT)(CO)4(CN)2]2−. Maturation with a [2Fe] complex in which the essential ADT amine moiety has been replaced by CH2 (PDT = propane-dithiolate) results in a low activity enzyme with structural and spectroscopic properties similar to those of the native enzyme, but with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 [Fe2(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.Electronic supplementary materialThe online version of this article (10.1007/s00775-018-1558-4) contains supplementary material, which is available to authorized users.

Geometry of the Catalytic Active Site in [FeFe]-Hydrogenase Is Determined by Hydrogen Bonding and Proton Transfer

“…12 Any variation of the cysteine or nitrogen head group severely diminishes H 2 turnover, which revealed the essential role of the ADT group as a proton transfer relay. [13][14][15] states Hox-CO and Hred´-CO suggests an additional, equatorial CO ligand and an apical cyanide ligand (aCN -) at Fe d although earlier crystal structures were modeled with an apical carbonyl (aCO). 20,21 Based on isotope editing studies we proposed aCO ligation at the reduced diiron site of Hred and Hsred together with a bridging hydride (µH -).…”

The Geometry of the Catalytic Active Site in [FeFe]-Hydrogenases is Determined by Hydrogen Bonding and Proton Transfer

“…S12). 14,15 ODT and EDT revealed a large population of Hred´ and Hhyd already at pH 8. 28 The EDT-modified enzyme showed an immediate Hox < Hred´ conversion followed by progressive enrichment of Hhyd over Hred´ (bottom left).…”

“…However, the reactivation kinetics indicate a higher degree of complexity. This possibly includes electrostatic attraction (ODT, SDT) 14 , steric repulsion between the apical ligand and the dithiolate head group (PDT) 15 , as well as differences in electron density distribution across the diiron site (PDS). 33 A notable spectroscopic feature is the lack of larger changes in the CNfrequencies among the COinhibited cofactor variants ( Fig.…”

“…12 Any variation of the cysteine or nitrogen head group severely diminishes H 2 turnover, which revealed the essential role of the ADT group as a proton transfer relay. [13][14][15] 5 states Hox-CO and Hred´-CO suggests an additional, equatorial CO ligand and an apical cyanide ligand (aCN -) at Fe d 19 although earlier crystal structures were modeled with an apical carbonyl (aCO). 20,21 Based on isotope editing studies we proposed aCO ligation at the reduced diiron site of Hred and Hsred together with a bridging hydride (µH -).…”

“…[31][32][33] Such cofactor variants show characteristic infrared signatures, accumulate different H-cluster states in the presence of 6 H 2 , and exhibit distinct CO sensitivities. [13][14][15] These observations prompted us to investigate the underlying substrate affinities and cofactor geometries. Nine variants of the [FeFe]hydrogenase from Chlamydomonas reinhardtii (HYDA1) were compared, including different dithiolate head groups as well as replacements of an adjacent cysteine (C169 in HYDA1) by side-directed mutagenesis.…”