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ABSTRACT: Non-exponential distance-dependence of the apparent electron transfer (ET) rate has been reported for a variety of redox proteins immobilized on biocompatible electrodes, thus posing a physicochemical challenge of possible physiological relevance. We have recently proposed that this behaviour may arise from the structural and dynamical complexity not only of the redox proteins, but also from their interplay with strong electric fields present in the experimental setups and in vivo (J. Am Chem. Soc 2010, 132, 5769-5778). Therefore, protein dynamics are finely controlled by the energetics of both specific contacts and the interaction between the protein's dipole moment and the interfacial electric fields. In turn, protein dynamics may govern electron transfer kinetics through reorientation from low to high donor-acceptor electronic coupling orientations. Here we present a combined computational and experimental study of WT cytochrome c and the surface mutant K87C adsorbed on electrodes coated with self assembled monolayers (SAMs) of varying thickness (i.e., variable strength of the interfacial electric field). Replacement of the positively charged K87 by a neutral amino acid allowed us to disentangle protein dynamics and electron tunnelling from the reaction kinetics and to rationalize the anomalous distance dependence in terms of (at least) two populations of distinct average electronic couplings. Thus, it was possible to recover the exponential distance dependence expected from ET theory. These results pave the way for gaining further insight into the parameters that control protein electron transfer.
The Journal of Physical Chemistry B 04/2013; · 3.70 Impact Factor
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Elisabeth Siebert,
Marius Horch,
Yvonne Rippers,
Johannes Fritsch,
Stefan Frielingsdorf,
Oliver Lenz,
Francisco Velazquez Escobar,
Friedrich Siebert,
Lars Paasche,
Uwe Kuhlmann,
Friedhelm Lendzian,
Maria-Andrea Mroginski,
Ingo Zebger, Peter Hildebrandt
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ABSTRACT: Insights in active sites: Hydrogen-conversion by hydrogenase is mediated by a sophisticated, metal-containing catalytic center. Resonance Raman spectroscopy is used for the first time in the characterization of the active site of these biocatalysts. An integrated spectroscopic and computational approach gives insights into structural and photochemical properties of the active site of an oxygen-tolerant [NiFe] hydrogenase.
Angewandte Chemie International Edition 04/2013; · 13.45 Impact Factor
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Johannes Salewski,
Francisco Velazquez Escobar,
Steve Kaminski,
David von Stetten,
Anke Keidel,
Yvonne Rippers,
Norbert Michael,
Patrick Scheerer,
Patrick Piwowarski,
Franz Bartl,
Nicole Frankenberg-Dinkel,
Simone Ringsdorf,
Wolfgang Gaertner,
Tilman Lamparter,
Maria Andrea Mroginski, Peter Hildebrandt
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ABSTRACT: The structures of the chromophore binding pockets in the Pfr states of various bathy and prototypical biliverdin-binding phytochromes were analysed by using a combined spectroscopic-theoretical approach. For the Pfr state of the bathy phytochrome from Pseudomonas aeruginosa (PaBphP) the very good agreement between calculated Raman spectra of the tetrapyrrole cofactor, obtained by quantum-mechanical / molecular-mechanical hybrid methods, and the experimental resonance Raman (RR) spectra confirms important conclusions derived from the previous crystallographic analyses, particularly the ZZEssa configuration of the chromophore and its attachment to the thiol side chain of Cys12 via the exocyclic vinyl group of ring A. The match between the RR spectra of the Pfr states of PaBphP and the bathy phytochrome Agp2 from Agrobacterium tumefaciens indicates very similar structures of the chromophore binding pockets. The homogeneous chromophore conformation in bathy phytochromes is in sharp contrast to the Pfr states of prototypical phytochromes as demonstrated by comparative RR spectroscopic analyses. The Pfr states of prototypical phytochromes, thoroughly studied for Agp1 (A. tumefaciens), display conformational equilibria between two sub-states differing with respect to the CD methine bridge torsional angle and the AB methine bridge geometry. These differences may mainly root in the interactions of the cofactor with the highly conserved Asp194 (PaBphP) that occur via its carboxylate function in bathy phytochromes. The weaker interactions via the carbonyl function in prototypical phytochromes may lead to a higher structural flexibility of the chromophore binding pocket that opens the reaction channel for the thermal (ZZE → ZZZ) Pfr-to-Pr back-conversion.
Journal of Biological Chemistry 04/2013; · 4.77 Impact Factor
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Subrata Kundu,
Florian Felix Pfaff,
Enrico Miceli,
Ivelina Zaharieva,
Christian Herwig,
Shenglai Yao,
Erik R Farquhar,
Uwe Kuhlmann,
Eckhard Bill, Peter Hildebrandt,
Holger Dau,
Matthias Driess,
Christian Limberg,
Kallol Ray
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ABSTRACT: Cores and effect: Unlike homobimetallic analogues, the heterobimetallic CuNi bis(μ-oxo) diamond core has nucleophilic oxo groups. A similar heterobimetallic core may, therefore, act as a viable intermediate during the deformylation of fatty aldehydes by cyanobacterial aldehyde decarbonylase.
Angewandte Chemie International Edition 04/2013; 125(21):5732-5736. · 13.45 Impact Factor
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Maria Andrea Mroginski,
David von Stetten,
Francisco Velazquez Escobar,
Holger M Strauss,
Steve Kaminski,
Patrick Scheerer,
Mina Günther,
Daniel H Murgida,
Peter Schmieder,
Christian Bongards,
Wolfgang Gärtner,
Jo Mailliet,
Jon Hughes,
Lars-Oliver Essen, Peter Hildebrandt
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David von Stetten,
Sven Seibeck,
Norbert Michael,
Patrick Scheerer,
Maria Andrea Mroginski,
Daniel H Murgida,
Norbert Krauss,
Maarten P Heyn, Peter Hildebrandt,
Berthold Borucki,
Tilman Lamparter
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ABSTRACT: The electron transfer (ET) processes of electroactive microbial biofilms have been investigated by combining electrochemistry and time-resolved surface-enhanced resonance Raman (TR-SERR) spectroscopy. This experimental approach provides selective information on the ET process across the biofilm-electrode interface by monitoring the redox-state changes of heme cofactors in outer membrane cytochromes (OMCs) that are in close vicinity (i.e., within 7 nm) to the Ag working electrode. The rate constant for heterogeneous ET of the surface-confined OMCs (sc-OMCs) of a mixed culture derived electroactive microbial biofilm has been determined to be 0.03 s(-1) . In contrast, according to kinetic simulations the ET between sc-OMCs and their redox partners, embedded within the biofilm, is a much faster process with an estimated rate constant greater than 1.2 s(-1) . The slow rate of heterogeneous ET and the lack of high-spin species in the SERR spectra rule out the direct attachment of the sc-OMCs to the electrode surface.
ChemSusChem 02/2013; · 6.83 Impact Factor
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ABSTRACT: The Vibrational Stark effect is gaining popularity as a method for probing electric fields in proteins. In this work, we employ it to explain the effect of single charge mutations in Dehaloperoxidase-hemoglobin A (DHP A) on the kinetics of the enzyme. In a previous communication published in this journal (BBRC 2012, 420, 733-737) it has been shown that an increase in the overall negative charge of DHP A through mutation causes a decrease in its catalytic efficiency. Here, by labeling the protein with 4-mercaptobenzonitrile (MBN), a Stark probe molecule, we provide further evidence that the diffusion control of the catalytic process arises from the electrostatic repulsion between the enzyme and the negatively charged substrate. The linear correlation observed between the nitrile stretching frequency of the protein-bound MBN and the catalytic efficiency of the single-site mutants of the enzyme indicates that electrostatic interactions play a dominant role in determining the catalytic efficiency of DHP A.
Biochemical and Biophysical Research Communications 12/2012; · 2.48 Impact Factor
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ABSTRACT: Understanding the interaction and immobilization of [NiFe] hydrogenases on functionalized surfaces is important in the field of biotechnology and in particular for the development of biofuel cells. In this study, we investigated the adsorption behavior of the standard [NiFe] hydrogenase of D. gigas on amino-terminated alkanethiol self-assembled monolayers (SAMs) with different levels of protonation. Classical all-atom MD simulations revealed a strong correlation between the adsorption behavior and the level of ionization of the chemically-modified electrode surface. While the hydrogenase undergoes a weak, but stable initial adsorption process on SAMs with a low degree of protonation, a stronger immobilization is observable on highly ionized SAMs, affecting protein reorientation and conformation. These results were validated by complementary SEIRA measurements on the comparable [NiFe] standard hydrogenases from D. vulgaris Miyazaki F, and allowed in this way for a detailed insight into the adsorption mechanism at the atomic level.
Langmuir 12/2012; · 4.19 Impact Factor
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ABSTRACT: Hydrogenases catalyse the reversible cleavage of molecular hydrogen into protons and electrons. While most of these enzymes are inhibited under aerobic conditions, some hydrogenases are catalytically active even at ambient oxygen levels. In particular, the soluble [NiFe] hydrogenase from Ralstonia eutropha H16 couples reversible hydrogen cycling to the redox conversion of NAD(H). Its insensitivity towards oxygen has been formerly ascribed to the putative presence of additional cyanide ligands at the active site, which has been, however, discussed controversially. Based on quantum chemical calculations of model compounds, we demonstrate that spectroscopic consequences of the proposed non-standard set of inorganic ligands are in contradiction to the underlying experimental findings. In this way, the previous model for structure and function of this soluble hydrogenase is disproved on a fundamental level, thereby highlighting the efficiency of computational methods for the evaluation of experimentally derived mechanistic proposals.
ChemPhysChem 11/2012; · 3.41 Impact Factor
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ABSTRACT: Combined molecular dynamics (MD) and quantum mechanical/molecular mechanical (QM/MM) calculations were performed on the crystal structure of the reduced membrane-bound [NiFe] hydrogenase (MBH) from Ralstonia eutropha to determine the absolute configuration of the CO and the two CN(-) ligands bound to the active-site iron of the enzyme. For three models that include the CO ligand at different positions, often indistinguishable on the basis of the crystallographic data, we optimized the structures and calculated the ligand stretching frequencies. Comparison with the experimental IR data reveals that the CO ligand is in trans position to the substrate-binding site of the bimetallic [NiFe] cluster.
ChemPhysChem 09/2012; · 3.41 Impact Factor
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ABSTRACT: High-valent copper-nitrene intermediates have long been proposed to play a role in copper-catalyzed aziridination and amination reactions. However, such intermediates have eluded detection for decades, preventing the unambiguous assignments of mechanisms. Moreover, the electronic structure of the proposed copper-nitrene intermediates has also been controversially discussed in the literature. These mechanistic questions and controversy have provided tremendous motivation to probe the accessibility and reactivity of Cu(III)-NR/Cu(II)N(•)R species. In this paper, we report a breakthrough in this field that was achieved by trapping a transient copper-tosylnitrene species, 3-Sc, in the presence of scandium triflate. The sufficient stability of 3-Sc at -90 °C enabled its characterization with optical, resonance Raman, NMR, and X-ray absorption near-edge spectroscopies, which helped to establish its electronic structure as Cu(II)N(•)Ts (Ts = tosyl group) and not Cu(III)NTs. 3-Sc can initiate tosylamination of cyclohexane, thereby suggesting Cu(II)N(•)Ts cores as viable reactants in oxidation catalysis.
Journal of the American Chemical Society 08/2012; 134(36):14710-3. · 9.91 Impact Factor
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ABSTRACT: Activation of the corrinoid [Fe-S] protein (CoFeSP), involved in reductive CO(2) conversion, requires the reduction of the Co(II) center by the [Fe-S] protein RACo, which according to the reduction potentials of the two proteins would correspond to an uphill electron transfer. In our resonance Raman spectroscopic work, we demonstrate that, as a conformational gate for the corrinoid reduction, complex formation of Co(II)FeSP and RACo specifically alters the structure of the corrinoid cofactor by modifying the interactions of the Co(II) center with the axial ligand. On the basis of various deletion mutants, the potential interaction domains on the partner proteins can be predicted.
Biochemistry 08/2012; 51(36):7040-2. · 3.42 Impact Factor
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ABSTRACT: Post-translational nitration of tyrosine is considered to be an important step in controlling the multiple functions of cytochrome c (Cyt-c). However, the underlying structural basis and mechanism are not yet understood. In this work, human Cyt-c variants in which all but one tyrosine has been substituted by phenylalanine have been studied by resonance Raman and electrochemical methods to probe the consequences of tyrosine nitration on the heme pocket structure and the redox potential. The mutagenic modifications of the protein cause only subtle conformational changes of the protein and small negative shifts of the redox potentials which can be rationalized in terms of long-range electrostatic effects on the heme. The data indicate that the contributions of the individual tyrosines for maintaining the relatively high redox potential of Cyt-c are additive. Nitration of individual tyrosines leads to a destabilization of the axial Fe-Met80 bond which causes the substitution of the native Met ligand by a water molecule or a lysine residue for a fraction of the proteins. Electrostatic immobilization of the protein variants on electrodes coated by self-assembled monolayers (SAMs) of mercaptounadecanoic acid destabilizes the heme pocket structure of both the nitrated and non-nitrated variants. Here, the involvement of surface lysines in binding to the SAM surface prevents the replacement of the Met80 ligand by a lysine but instead a His-His coordinated species is formed. The results indicate that structural perturbations of the heme pocket of Cyt-c due to tyrosine nitration and to local electric fields are independent of each other and occur via different molecular mechanisms. The present results are consistent with the view that either tyrosine nitration or electrostatic binding to the inner mitochondrial membrane, or both events together, are responsible for the switch from the redox to the peroxidase function.
The Journal of Physical Chemistry B 04/2012; · 3.70 Impact Factor
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ABSTRACT: Analyzing the structure and function of redox enzymes attached to electrodes is a central challenge in many fields of fundamental and applied life science. Electrochemical techniques such as cyclic voltammetry which are routinely used do not provide insight into the molecular structure and reaction mechanisms of the immobilized proteins. Surface-enhanced infrared absorption (SEIRA) and surface-enhanced resonance Raman (SERR) spectroscopy may fill this gap, if nanostructured Au or Ag are used as conductive support materials. In this account, we will first outline the principles of the methodology including a description of the most important strategies for biocompatible protein immobilization. Subsequently, we will critically review SERR and SEIRA spectroscopic approaches to characterize the protein and active site structure of the immobilized enzymes. Special emphasis is laid on the combination of surface-enhanced vibrational spectroscopies with electrochemical methods to analyze equilibria and dynamics of the interfacial redox processes. Finally, we will assess the potential of SERR and SEIRA spectroscopy for in situ investigations on the basis of the first promising studies on human sulfite oxidase and hydrogenases under turnover conditions.
International Union of Biochemistry and Molecular Biology Life 04/2012; 64(6):455-64. · 3.51 Impact Factor
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ABSTRACT: 4-mercaptobenzonitrile (MBN) in self-assembled monolayers (SAMs) on Au and Ag electrodes was studied by surface enhanced infrared absorption and Raman spectroscopy, to correlate the nitrile stretching frequency with the local electric field exploiting the vibrational Stark effect (VSE). Using MBN SAMs in different metal/SAM interfaces, we sorted out the main factors controlling the nitrile stretching frequency, which comprise, in addition to external electric fields, the metal-MBN bond, the surface potential, and hydrogen bond interactions. On the basis of the linear relationships between the nitrile stretching and the electrode potential, an electrostatic description of the interfacial potential distribution is presented that allows for determining the electric field strengths on the SAM surface, as well as the effective potential of zero-charge of the SAM-coated metal. Comparing this latter quantity with calculated values derived from literature data, we note a very good agreement for Au/MBN but distinct deviations for Ag/MBN which may reflect either the approximations and simplifications of the model or the uncertainty in reported structural parameters for Ag/MBN. The present electrostatic model consistently explains the electric field strengths for MBN SAMs on Ag and Au as well as for thiophenol and mercaptohexanoic acid SAMs with MBN incorporated as a VSE reporter.
International Journal of Molecular Sciences 01/2012; 13(6):7466-82. · 2.60 Impact Factor
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ABSTRACT: The photocycle of the light-activated channel, channelrhodopsin-2 C128T, has been studied by resonance Raman (RR) spectroscopy focussing on the intermediates P380 and P353 that constitute a side pathway in the recovery of the parent state. The P353 species displays a UV-vis absorption spectrum with a fine-structure reminiscent of the reduced-retro form of bacteriorhodopsin, whereas the respective RR spectra differ substantially. Instead, the RR spectra of the P380/P353 intermediate couple are closely related to that of a free retinal in the all-trans configuration. These findings imply that the parent state recovery via P380/P353 involves the transient hydrolysis and re-formation of the retinal-protein linkage.
FEBS letters 11/2011; 585(24):3998-4001. · 3.54 Impact Factor
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Gal Schkolnik,
Tillmann Utesch,
Johannes Salewski,
Katalin Tenger,
Diego Millo,
Anja Kranich,
Ingo Zebger,
Claudia Schulz,
László Zimányi,
Gábor Rákhely,
Maria Andrea Mroginski, Peter Hildebrandt
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ABSTRACT: We present a novel approach for determining the strength of the electric field experienced by proteins immobilised on membrane models. It is based on the vibrational Stark effect of a nitrile label introduced at different positions on engineered proteins and monitored by surface enhanced infrared absorption spectroscopy.
Chemical Communications 11/2011; 48(1):70-2. · 6.17 Impact Factor
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ABSTRACT: The role of the diheme cytochrome b (HoxZ) subunit in the electron transfer pathway of the membrane-bound [NiFe]-hydrogenase (MBH) heterotrimer from Ralstonia eutropha H16 has been investigated. The MBH in its native heterotrimeric state was immobilized on electrodes and subjected to spectroscopic and electrochemical analysis. Surface enhanced resonance Raman spectroscopy was used to monitor the redox and coordination state of the HoxZ heme cofactors while concomitant protein film voltammetric measurements gave insights into the catalytic response of the enzyme on the electrode. The entire MBH heterotrimer as well as its isolated HoxZ subunit were immobilized on silver electrodes coated with self-assembled monolayers of ω-functionalized alkylthiols, displaying the preservation of the native heme pocket structure and an electrical communication between HoxZ and the electrode. For the immobilized MBH heterotrimer, catalytic reduction of the HoxZ heme cofactors was observed upon H(2) addition. The catalytic currents of MBH with and without the HoxZ subunit were measured and compared with the heterogeneous electron transfer rates of the isolated HoxZ. On the basis of the spectroscopic and electrochemical results, we conclude that the HoxZ subunit under these artificial conditions is not primarily involved in the electron transfer to the electrode but plays a crucial role in stabilizing the enzyme on the electrode.
The Journal of Physical Chemistry B 09/2011; 115(34):10368-74. · 3.70 Impact Factor
