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

JBIC Journal of Biological Inorganic Chemistry, Apr 2018

[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 Maturation with a [2Fe] complex in which the essential ADT amine moiety has been replaced by CH2 (PDT = propane-dithiolate) results in a low activity enzyme with structural and spectroscopic properties similar to those of the native enzyme, but with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.

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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

JBIC Journal of Biological Inorganic Chemistry May 2018, Volume 23, Issue 3, pp 481–491 | Cite as 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 AuthorsAuthors and affiliations C. SommerS. RumpelS. RoyC. FarèsV. ArteroM. FontecaveE. ReijerseW. Lubitz Open Access Original Paper First Online: 07 April 2018 1 Shares 1.3k Downloads 4 Citations 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. Keywords[FeFe] Hydrogenase Chalcogenic substitution Nuclear magnetic resonance Electron paramagnetic resonance FTIR spectroelectrochemistry  Abbreviations MV Methyl viologen NMR Nuclear magnetic resonance EPR Electron paramagnetic resonance FTIR Fourier transform infrared spectroscopy Bridging ligands ADT Aza-propane-dithiolate (µ(SCH2)2NH) ADSe Aza-propane-diselenate (µ(SeCH2)2NH) PDT Propane-dithiolate (µ(SCH2)2CH2) PDSe Propane-diselenate (µ(SeCH2)2CH2) Synthetic precursors [2Fe]–ADT/–ADSe/–PDT/–PDSe [2Fe] = Fe2(CO)4(CN−)2 Enzymes apo-HydA1 CrHydA1 containing only the [4Fe–4S/Se]H subsite HydA1-ADT/-PDT CrHydA1 maturated with [2Fe]-ADT or [2Fe]-PDT Cr Chlamydomonas reinhardtii Subsites [2Fe]H Fe2(CO)3(CN−)2ADT/ADSe/PDT/PDSe, subsite of [FeFe] hydrogenase Electronic supplementary material The online version of this article ( https://doi.org/10.1007/s00775-018-1558-4) contains supplementary material, which is available to authorized users. Introduction The reversible heterolytic splitting of hydrogen into protons and electrons is one of the most fundamental reactions in chemistry. In nature, hydrogen is part of the energy metabolism of several single cellular organisms which are spread over all three domains of life [1, 2]. [FeFe] Hydrogenases catalyze the conversion of protons and electrons into hydrogen in a very efficient way with turnover frequencies over 10,000 H2/s [3, 4]. The active site in these enzymes, the so-called H-cluster, consists of a generic [4Fe–4S]H cluster linked to a binuclear iron complex [2Fe]H carrying 3 CO and 2 CN− ligands as well as a bridging aza-propane-dithiolate (ADT) ligand (see Fig. 1) that serves as proton relay of the protein’s proton transport pathway [5]. Often, additional [4Fe–4S] clusters are present that form an electron transport chain connecting the H-cluster with the protein surface where redox partners of the enzyme can bind. Open image in new window Fig. 1 The native active site of [FeFe] hydrogenase and applied modifications. The iron atoms are labeled as proximal (Fep) and distal (Fed) with respect to their position to the [4Fe–4S]H cluster. Left: native H-cluster that consists of the [4Fe–4S]H cluster and [2Fe]H-ADT subsite. Right: modified H-cluster with [2Fe]H-PDT or [2Fe]H-PDSe. Additionally bridging sulfides in the cubane cluster (here marked with X) can be exchanged to Se The small hydrogenase from Chlamydomonas reinhardtii HydA1 is used as prototype for [FeFe] hydrogenases, since it contains only the H-cluster and can be overexpressed in Escherichia coli [6] with high yields [7]. However, since the host organism lacks the maturation factors that built the (...truncated)


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C. Sommer, S. Rumpel, S. Roy, C. Farès, V. Artero, M. Fontecave, E. Reijerse, W. Lubitz. 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, JBIC Journal of Biological Inorganic Chemistry, 2018, pp. 481-491, Volume 23, Issue 3, DOI: 10.1007/s00775-018-1558-4