[NiFe]-hydrogenases catalyze the reversible activation of H using a unique NiFe(CN)CO metal site, which is assembled by a sophisticated multiprotein machinery. The [4Fe-4S] cluster-containing HypCD complex, which possesses an ATPase activity with a hitherto unknown function, serves as the hub for the assembly of the Fe(CN)CO subfragment. HypCD is also thought to be responsible for the subsequent transfer of the iron fragment to the apo-form of the catalytic hydrogenase subunit, but the underlying mechanism has remained unexplored.
View Article and Find Full Text PDFNickel complexes with a two-electron reduced CO ligand (CO , "carbonite") are investigated with regard to the influence alkali metal (AM) ions have as Lewis acids on the activation of the CO entity. For this purpose complexes with Ni(CO)AM (AM=Li, Na, K) moieties were accessed via deprotonation of nickel-formate compounds with (AM)N(Pr). It was found that not only the nature of the AM ions in vicinity to CO affect the activation, but also the number and the ligation of a given AM.
View Article and Find Full Text PDFContinued efforts are made on the development of earth-abundant metal catalysts for dehydrogenation/hydrolysis of amine boranes. In this study, complex [K-18-crown-6-ether][(NO)Fe(μ-Pyr)(μ-CO)Fe(NO)] (, Pyr = 3-methylpyrazolate) was explored as a pre-catalyst for the dehydrogenation of dimethylamine borane (DMAB). Upon evolution of H from DMAB triggered by , parallel conversion of into [(NO)Fe(,'-PyrBHNMe)] () and an iron-hydride intermediate [(NO)(CO)Fe(μ-H)Fe(CO)(NO)] () was evidenced by X-ray diffraction/nuclear magnetic resonance/infrared/nuclear resonance vibrational spectroscopy experiments and supported by density functional theory calculations.
View Article and Find Full Text PDFThe noncubane [4Fe-4S] cluster identified in the active site of heterodisulfide reductase (HdrB) displays a unique geometry among Fe-S cofactors found in metalloproteins. Here we employ resonance Raman (RR) spectroscopy and density functional theory (DFT) calculations to probe structural, electronic, and vibrational properties of the noncubane cluster in HdrB from a non-methanogenic () Hildenborough organism. The immediate protein environment of the two neighboring clusters in HdrB is predicted using homology modeling.
View Article and Find Full Text PDFThe human mitochondrial protein, mitoNEET (mNT), belongs to the family of small [2Fe-2S] NEET proteins that bind their iron-sulfur clusters with a novel and characteristic 3Cys:1His coordination motif. mNT has been implicated in the regulation of lipid and glucose metabolisms, iron/reactive oxygen species homeostasis, cancer, and possibly Parkinson's disease. The geometric structure of mNT as a function of redox state and pH is critical for its function.
View Article and Find Full Text PDFThe catalytic mechanism of [NiFe]-hydrogenases is a subject of extensive research. Apart from at least four reaction intermediates of H/H cycling, there are also a number of resting states, which are formed under oxidizing conditions. Although not directly involved in the catalytic cycle, the knowledge of their molecular structures and reactivity is important, because these states usually accumulate in the course of hydrogenase purification and may also play a role during hydrogenase maturation.
View Article and Find Full Text PDFA diiron complex containing a bridging hydride and a protonated terminal thiolate of the form [(μ,κ-bdtH)(μ-PPh)(μ-H)Fe(CO)] has been investigated through Fe nuclear resonance vibrational spectroscopy (NRVS) and interpreted using density functional theory (DFT) calculations. We report the Fe-μH-Fe wagging mode, and indications for Fe-μD stretching vibrations in the D-isotopologue, observed by Fe-NRVS. Our combined approach demonstrates an asymmetric sharing of the hydride between the two iron sites that yields two nondegenerate Fe-μH/D stretching vibrations.
View Article and Find Full Text PDF[FeFe] hydrogenases are highly active catalysts for the interconversion of molecular hydrogen with protons and electrons. Here, we use a combination of isotopic labeling, Fe nuclear resonance vibrational spectroscopy (NRVS), and density functional theory (DFT) calculations to observe and characterize the vibrational modes involving motion of the 2-azapropane-1,3-dithiolate (ADT) ligand bridging the two iron sites in the [2Fe] subcluster. A -CH- ADT labeling in the synthetic diiron precursor of [2Fe] produced isotope effects observed throughout the NRVS spectrum.
View Article and Find Full Text PDFTo study metalloenzymes in detail, we developed a new experimental setup allowing the controlled preparation of catalytic intermediates for characterization by various spectroscopic techniques. The in situ monitoring of redox transitions by infrared spectroscopy in enzyme lyophilizate, crystals, and solution during gas exchange in a wide temperature range can be accomplished as well. Two O -tolerant [NiFe]-hydrogenases were investigated as model systems.
View Article and Find Full Text PDF[NiFe]-hydrogenases catalyze the reversible reaction H ⇄ 2H + 2e. Their basic module consists of a large subunit, coordinating the NiFe(CO)(CN) center, and a small subunit that carries electron-transferring iron-sulfur clusters. Here, we report the assembly of fully functional [NiFe]-hydrogenase starting from the isolated large and small subunits.
View Article and Find Full Text PDFNuclear resonance vibrational spectroscopy (NRVS) and density functional theory (DFT) are complementary tools for studying the vibrational and geometric structures of specific isotopically labeled molecular systems. Here we apply NRVS and DFT to characterize the -[Fe(η-H)(H)(dppe)][BPh] [dppe = 1,2-bis(diphenylphosphino)ethane] complex. Heretofore, most NRVS observations have centered on the spectral region below 1000 cm, where the Fe signal is strongest.
View Article and Find Full Text PDFMost of our understanding of chemistry derives from atomic-level structures obtained with single-crystal X-ray diffraction. Metal centers in X-ray structures of small organometallic or coordination complexes are often extremely well-defined, with errors in the positions on the order of 10-10 Å. Determining the metal coordination geometry to high accuracy is essential for understanding metal center reactivity, as even small structural changes can dramatically alter the metal activity.
View Article and Find Full Text PDF[FeFe] hydrogenases are extremely active H-converting enzymes. Their mechanism remains highly controversial, in particular, the nature of the one-electron and two-electron reduced intermediates called HH and HH. In one model, the HH and HH states contain a semibridging CO, while in the other model, the bridging CO is replaced by a bridging hydride.
View Article and Find Full Text PDFIron-sulfur clusters are common building blocks for electron transport and active sites of metalloproteins. Their comprehensive investigation is crucial for understanding these enzymes, which play important roles in modern biomimetic catalysis and biotechnology applications. We address this issue by utilizing (EtN)[FeTe(SPh)], a tellurium modified version of a conventional reduced [4Fe-4S] cluster, and performed both Fe- and Te-NRVS to reveal its characteristic vibrational features.
View Article and Find Full Text PDF[FeFe] hydrogenases are very active enzymes that catalyze the reversible conversion of molecular hydrogen into protons and electrons. Their active site, the H-cluster, contains a unique binuclear iron complex, [2Fe], with CN and CO ligands as well as an aza-propane-dithiolate (ADT) moiety featuring a central amine functionality that mediates proton transfer during catalysis. We present a pulsed C-ENDOR investigation of the H-cluster in which the two methylene carbons of ADT are isotope labeled with C.
View Article and Find Full Text PDFA combination of nuclear resonance vibrational spectroscopy (NRVS), FTIR spectroscopy, and DFT calculations was used to observe and characterize Fe-H/D bending modes in CrHydA1 [FeFe]-hydrogenase Cys-to-Ser variant C169S. Mutagenesis of cysteine to serine at position 169 changes the functional group adjacent to the H-cluster from a -SH to -OH, thus altering the proton transfer pathway. The catalytic activity of C169S is significantly reduced compared to that of native CrHydA1, presumably owing to less efficient proton transfer to the H-cluster.
View Article and Find Full Text PDFHigh-spin iron species with bridging hydrides have been detected in species trapped during nitrogenase catalysis, but there are few general methods of evaluating Fe-H bonds in high-spin multinuclear iron systems. An Fe nuclear resonance vibrational spectroscopy (NRVS) study on an Fe(μ-H) Fe model complex reveals Fe-H stretching vibrations for bridging hydrides at frequencies greater than 1200 cm . These isotope-sensitive vibrational bands are not evident in infrared (IR) spectra, showing the power of NRVS for identifying hydrides in this high-spin iron system.
View Article and Find Full Text PDFThe kinetically robust hydride [t-HFe(Mepdt)(CO)(dppv)] ([t-H1]) (Mepdt = MeC(CHS); dppv = cis-1,2-CH(PPh)) and related derivatives were prepared with Fe enrichment for characterization by NMR, FT-IR, and NRVS. The experimental results were rationalized using DFT molecular modeling and spectral simulations. The spectroscopic analysis was aimed at supporting assignments of Fe-H vibrational spectra as they relate to recent measurements on [FeFe]-hydrogenase enzymes.
View Article and Find Full Text PDF[FeFe]-hydrogenases are metalloenzymes that reversibly reduce protons to molecular hydrogen at exceptionally high rates. We have characterized the catalytically competent hydride state (H) in the [FeFe]-hydrogenases from both Chlamydomonas reinhardtii and Desulfovibrio desulfuricans using Fe nuclear resonance vibrational spectroscopy (NRVS) and density functional theory (DFT). H/D exchange identified two Fe-H bending modes originating from the binuclear iron cofactor.
View Article and Find Full Text PDF[FeFe]-hydrogenases catalyze the reversible reduction of protons to molecular hydrogen with extremely high efficiency. The active site ("H-cluster") consists of a [4Fe-4S] cluster linked through a bridging cysteine to a [2Fe] subsite coordinated by CN and CO ligands featuring a dithiol-amine moiety that serves as proton shuttle between the protein proton channel and the catalytic distal iron site (Fe). Although there is broad consensus that an iron-bound terminal hydride species must occur in the catalytic mechanism, such a species has never been directly observed experimentally.
View Article and Find Full Text PDFAn unprecedented [4Fe-3S] cluster proximal to the regular [NiFe] active site has recently been found to be responsible for the ability of membrane-bound hydrogenases (MBHs) to oxidize dihydrogen in the presence of ambient levels of oxygen. Starting from proximal cluster models of a recent DFT study on the redox-dependent structural transformation of the [4Fe-3S] cluster, (57)Fe Mössbauer parameters (electric field gradients, isomer shifts, and nuclear hyperfine couplings) were calculated using DFT. Our results revise the previously reported correspondence of Mössbauer signals and iron centers in the [4Fe-3S](3+) reduced-state proximal cluster.
View Article and Find Full Text PDFWe have used femtosecond pump-probe spectroscopy (FPPS) to study the FeMo-cofactor within the nitrogenase (N2ase) MoFe protein from Azotobacter vinelandii. A sub-20-fs visible laser pulse was used to pump the sample to an excited electronic state, and a second sub-10-fs pulse was used to probe changes in transmission as a function of probe wavelength and delay time. The excited protein relaxes to the ground state with a ~1.
View Article and Find Full Text PDFThe metabolism of many anaerobes relies on [NiFe]-hydrogenases, whose characterization when bound to substrates has proven non-trivial. Presented here is direct evidence for a hydride bridge in the active site of the (57)Fe-labelled fully reduced Ni-R form of Desulfovibrio vulgaris Miyazaki F [NiFe]-hydrogenase. A unique 'wagging' mode involving H(-) motion perpendicular to the Ni(μ-H)(57)Fe plane was studied using (57)Fe-specific nuclear resonance vibrational spectroscopy and density functional theory (DFT) calculations.
View Article and Find Full Text PDFJ Phys Chem B
October 2015
We have applied resonance Raman (RR) spectroscopy on single protein crystals of the O2-tolerant membrane-bound [NiFe] hydrogenase (MBH from Ralstonia eutropha) which catalyzes the splitting of H2 into protons and electrons. RR spectra taken from 65 MBH samples in different redox states were analyzed in terms of the respective component spectra of the active site and the unprecedented proximal [4Fe-3S] cluster using a combination of statistical methods and global fitting procedures. These component spectra of the individual cofactors were compared with calculated spectra obtained by quantum mechanics/molecular mechanics (QM/MM) methods.
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