ABSTRACT [FeFe]-hydrogenases catalyze the reversible interconversion of protons to molecular hydr... more ABSTRACT [FeFe]-hydrogenases catalyze the reversible interconversion of protons to molecular hydrogen (H2) at an active site called H-cluster. The maturation pathway of these enzymes is a complex process involving three proteins, HydE, HydF and HydG. The maturase protein HydF has been suggested to interact with HydE and HydG and to be the transferase that shuttles the complete H-cluster to the hydrogenase; however, the exact molecular mechanism driving this translocation remains unclear. HydF is constituted by three different domains: a N-terminal GTP-binding domain, a dimerization domain and a C-terminal [4Fe4S] cluster-binding domain. To investigate possible conformational changes induced by the GTP binding in the N-terminal domain, we have expressed, in Escherichia coli, a recombinant HydF protein from Thermotoga neapolitana including the GTP-binding domain only. Site-directed mutants were designed in which the native residues were substituted by cysteines and subsequently spin labeled with the nitroxide MTSSL. CW-EPR was used to study the local mobility of the nitroxides at each site, and double spin-labeled mutants have been investigated by PELDOR spectroscopy. We found that the binding of the nucleotide does not induce large conformational effects within the isolated GTP domain, at least at the level of the elements investigated in this work. However, small changes in the distance between spin labels were observed which might reflect diffuse structural rearrangements. We suggest that the variations following the GTP binding could affect the dimer form adopted by the whole HydF protein in solution and, as a consequence, the interactions with the other maturases.
This work demonstrates, for the first time, the feasibility of applying pulsed electron-electron ... more This work demonstrates, for the first time, the feasibility of applying pulsed electron-electron double resonance (PELDOR/DEER) to determine the interspin distance between a photoexcited porphyrin triplet state (S = 1) and a nitroxide spin label chemically incorporated into a small helical peptide. The PELDOR trace shows deep envelope modulation induced by electron-electron dipole interaction between the partners in the pair, providing an accurate distance measurement. This new labeling approach has a high potential for measuring nanometer distances in more complex biological systems due to the sensitivity acquired from the spin polarization of the photoexcited triplet state spectrum.
ABSTRACT [FeFe]-hydrogenases contain a complex [4Fe–4S]-2Fe cluster (H-cluster) and are able to e... more ABSTRACT [FeFe]-hydrogenases contain a complex [4Fe–4S]-2Fe cluster (H-cluster) and are able to efficiently reduce protons to H2. Due to their potential exploitation for renewable energy production biotechnologies, significant efforts have been put into understanding the mechanisms driving the H-cluster assembly, which involves three conserved proteins. Among them, HydF works as scaffold upon which the H-cluster precursor is synthesized and carrier to deliver it to the hydrogenase, resulting in its activation. A FeS cluster binding sequence (CxHx46-53HCxxC) is conserved in all HydF proteins and should in principle provide four ligands to coordinate the Fe atom. However, we found that alternative metal coordination may exist in different HydF proteins and that only the three cysteines are strictly required, whereas the fourth ligand may vary and is, in any case, readily exchangeable. In this work we analyzed by EPR/HYSCORE the FeS cluster proton environment of HydF from Thermotoga neapolitana to determine the possible role of surrounding residues in the non-cysteinyl iron ligation of the protein.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2012
[FeFe] hydrogenases are key enzymes for bio(photo)production of molecular hydrogen, and several e... more [FeFe] hydrogenases are key enzymes for bio(photo)production of molecular hydrogen, and several efforts are underway to understand how their complex active site is assembled. This site contains a [4Fe-4S]-2Fe cluster and three conserved maturation proteins are required for its biosynthesis. Among them, HydF has a double task of scaffold, in which the dinuclear iron precursor is chemically modified by the two other maturases, and carrier to transfer this unit to a hydrogenase containing a preformed [4Fe-4S]-cluster. This dual role is associated with the capability of HydF to bind and dissociate an iron-sulfur center, due to the presence of the conserved FeS-cluster binding sequence CxHx(46-53)HCxxC. The recently solved three-dimensional structure of HydF from Thermotoga neapolitana described the domain containing the three cysteines which are supposed to bind the FeS cluster, and identified the position of two conserved histidines which could provide the fourth iron ligand. The functional role of two of these cysteines in the activation of [FeFe]-hydrogenases has been confirmed by site-specific mutagenesis. On the other hand, the contribution of the three cysteines to the FeS cluster coordination sphere is still to be demonstrated. Furthermore, the potential role of the two histidines in [FeFe]-hydrogenase maturation has never been addressed, and their involvement as fourth ligand for the cluster coordination is controversial. In this work we combined site-specific mutagenesis with EPR (electron paramagnetic resonance) and HYSCORE (hyperfine sublevel correlation spectroscopy) to assign a role to these conserved residues, in both cluster coordination and hydrogenase maturation/activation, in HydF proteins from different microorganisms.
The journal of physical chemistry. B, Jan 27, 2015
The catalytic site of [FeFe]-hydrogenase, the "H-cluster", composed of a [4Fe-4S] unit ... more The catalytic site of [FeFe]-hydrogenase, the "H-cluster", composed of a [4Fe-4S] unit connected by a cysteinyl residue to a [2Fe] center coordinated by three CO, two CN(-), and a bridging dithiolate, is assembled in a complex maturation pathway, at present not fully characterized, involving three conserved proteins, HydG, HydE, and HydF. HydF is a complex enzyme, which is thought to act as a scaffold and carrier for the [2Fe] subunit of the H-cluster. This maturase protein contains itself a [4Fe-4S] cluster binding site, with three conserved cysteine residues and a noncysteinyl fourth ligand. In this work, we have exploited 3p-ESEEM and HYSCORE spectroscopies to get insight into the structure and the chemical environment of the [4Fe-4S] cluster of HydF from the hyperthermophilic organism Thermotoga neapolitana. The nature of the fourth ligand and the solvent accessibility of the active site comprising the [4Fe-4S] cluster are discussed on the basis of the spectroscopic re...
ABSTRACT [FeFe]-hydrogenases catalyze the reversible interconversion of protons to molecular hydr... more ABSTRACT [FeFe]-hydrogenases catalyze the reversible interconversion of protons to molecular hydrogen (H2) at an active site called H-cluster. The maturation pathway of these enzymes is a complex process involving three proteins, HydE, HydF and HydG. The maturase protein HydF has been suggested to interact with HydE and HydG and to be the transferase that shuttles the complete H-cluster to the hydrogenase; however, the exact molecular mechanism driving this translocation remains unclear. HydF is constituted by three different domains: a N-terminal GTP-binding domain, a dimerization domain and a C-terminal [4Fe4S] cluster-binding domain. To investigate possible conformational changes induced by the GTP binding in the N-terminal domain, we have expressed, in Escherichia coli, a recombinant HydF protein from Thermotoga neapolitana including the GTP-binding domain only. Site-directed mutants were designed in which the native residues were substituted by cysteines and subsequently spin labeled with the nitroxide MTSSL. CW-EPR was used to study the local mobility of the nitroxides at each site, and double spin-labeled mutants have been investigated by PELDOR spectroscopy. We found that the binding of the nucleotide does not induce large conformational effects within the isolated GTP domain, at least at the level of the elements investigated in this work. However, small changes in the distance between spin labels were observed which might reflect diffuse structural rearrangements. We suggest that the variations following the GTP binding could affect the dimer form adopted by the whole HydF protein in solution and, as a consequence, the interactions with the other maturases.
This work demonstrates, for the first time, the feasibility of applying pulsed electron-electron ... more This work demonstrates, for the first time, the feasibility of applying pulsed electron-electron double resonance (PELDOR/DEER) to determine the interspin distance between a photoexcited porphyrin triplet state (S = 1) and a nitroxide spin label chemically incorporated into a small helical peptide. The PELDOR trace shows deep envelope modulation induced by electron-electron dipole interaction between the partners in the pair, providing an accurate distance measurement. This new labeling approach has a high potential for measuring nanometer distances in more complex biological systems due to the sensitivity acquired from the spin polarization of the photoexcited triplet state spectrum.
ABSTRACT [FeFe]-hydrogenases contain a complex [4Fe–4S]-2Fe cluster (H-cluster) and are able to e... more ABSTRACT [FeFe]-hydrogenases contain a complex [4Fe–4S]-2Fe cluster (H-cluster) and are able to efficiently reduce protons to H2. Due to their potential exploitation for renewable energy production biotechnologies, significant efforts have been put into understanding the mechanisms driving the H-cluster assembly, which involves three conserved proteins. Among them, HydF works as scaffold upon which the H-cluster precursor is synthesized and carrier to deliver it to the hydrogenase, resulting in its activation. A FeS cluster binding sequence (CxHx46-53HCxxC) is conserved in all HydF proteins and should in principle provide four ligands to coordinate the Fe atom. However, we found that alternative metal coordination may exist in different HydF proteins and that only the three cysteines are strictly required, whereas the fourth ligand may vary and is, in any case, readily exchangeable. In this work we analyzed by EPR/HYSCORE the FeS cluster proton environment of HydF from Thermotoga neapolitana to determine the possible role of surrounding residues in the non-cysteinyl iron ligation of the protein.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2012
[FeFe] hydrogenases are key enzymes for bio(photo)production of molecular hydrogen, and several e... more [FeFe] hydrogenases are key enzymes for bio(photo)production of molecular hydrogen, and several efforts are underway to understand how their complex active site is assembled. This site contains a [4Fe-4S]-2Fe cluster and three conserved maturation proteins are required for its biosynthesis. Among them, HydF has a double task of scaffold, in which the dinuclear iron precursor is chemically modified by the two other maturases, and carrier to transfer this unit to a hydrogenase containing a preformed [4Fe-4S]-cluster. This dual role is associated with the capability of HydF to bind and dissociate an iron-sulfur center, due to the presence of the conserved FeS-cluster binding sequence CxHx(46-53)HCxxC. The recently solved three-dimensional structure of HydF from Thermotoga neapolitana described the domain containing the three cysteines which are supposed to bind the FeS cluster, and identified the position of two conserved histidines which could provide the fourth iron ligand. The functional role of two of these cysteines in the activation of [FeFe]-hydrogenases has been confirmed by site-specific mutagenesis. On the other hand, the contribution of the three cysteines to the FeS cluster coordination sphere is still to be demonstrated. Furthermore, the potential role of the two histidines in [FeFe]-hydrogenase maturation has never been addressed, and their involvement as fourth ligand for the cluster coordination is controversial. In this work we combined site-specific mutagenesis with EPR (electron paramagnetic resonance) and HYSCORE (hyperfine sublevel correlation spectroscopy) to assign a role to these conserved residues, in both cluster coordination and hydrogenase maturation/activation, in HydF proteins from different microorganisms.
The journal of physical chemistry. B, Jan 27, 2015
The catalytic site of [FeFe]-hydrogenase, the "H-cluster", composed of a [4Fe-4S] unit ... more The catalytic site of [FeFe]-hydrogenase, the "H-cluster", composed of a [4Fe-4S] unit connected by a cysteinyl residue to a [2Fe] center coordinated by three CO, two CN(-), and a bridging dithiolate, is assembled in a complex maturation pathway, at present not fully characterized, involving three conserved proteins, HydG, HydE, and HydF. HydF is a complex enzyme, which is thought to act as a scaffold and carrier for the [2Fe] subunit of the H-cluster. This maturase protein contains itself a [4Fe-4S] cluster binding site, with three conserved cysteine residues and a noncysteinyl fourth ligand. In this work, we have exploited 3p-ESEEM and HYSCORE spectroscopies to get insight into the structure and the chemical environment of the [4Fe-4S] cluster of HydF from the hyperthermophilic organism Thermotoga neapolitana. The nature of the fourth ligand and the solvent accessibility of the active site comprising the [4Fe-4S] cluster are discussed on the basis of the spectroscopic re...
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Papers by Marco Albertini