156 research outputs found
The rates of proton uptake and electron transfer at the reducing side of photosystem II in thylakoids
AbstractProton and electron transfer at the reducing side of photosystem II of green plants was studied under flashing light, the former at improved time resolution by using Neutral red. The rates of electron transfer within QAFeQB were determined by pump-probe flashes through electrochromic transients. The extent of proton binding was about 1 H+/eâ. The rates of proton transfer were proportional to the concentration of Neutral red (collisional transfer), whereas the rates of electron transfer out of QâA and from QAFeQâB to the cytochrome b6f complex were constant. The half-rise times of electron transfer (Ïe) and the apparent times of proton binding (Ïh) at 30 ÎŒM Neutral red were: QâA â FeIIIQB (Ïe â©œ 100 ÎŒs, Ï, 230 ÎŒs); QâA â FeIIQB (Ïe = 150 ÎŒs, Ïh = 760 ÎŒs); and QâA â FeIIQâB (Ïe = 620 ÎŒs, Ïh = 310 ÎŒs)
Evidence for impaired hydrogen-bonding of tyrosine YZ in calcium-depleted Photosystem II
AbstractPhotosystem II (PS II) evolves oxygen from two bound water molecules in a four-stepped reaction that is driven by four quanta of light, each oxidizing the chlorophyll moiety P680 to yield P+680. When starting from its dark equilibrium (mainly state S1), the catalytic center can be clocked through its redox states (S0âŠS4) by a series of short flashes of light. The center involves at least a Mn4-cluster and a special tyrosine residue, named YZ, as redox cofactors plus two essential ionic cofactors, Clâ and Ca2+. Centers which have lost Ca2+ do not evolve oxygen. We investigated the stepped progression in dark-adapted PS II core particles after the removal of Ca2+. YZ was oxidized from the first flash on. The difference spectrum of YZâYoxZ differed from the one in competent centers, where it has been ascribed to a hydrogen-bonded tyrosinate. The rate of the electron transfer from YZ to P+680 was slowed down by three orders of magnitude and its kinetic isotope effect rose up from 1.1 to 2.5. Proton release into the bulk was now a prerequisite for the electron transfer from YZ to P+680. On the basis of these results and similar effects in Mn-(plus Ca2+-)depleted PS II (M. Haumann et al., Biochemistry, 38 (1999) 1258â1267) we conclude that the presence of Ca2+ in the catalytic center is required to tune the apparent pK of a base cluster, B, to which YZ is linked by hydrogen bonds. The deposition of a proton on B within close proximity of YZ (not its release into the bulk!) is a necessary condition for the reduction in nanoseconds of P+680 and for the functioning of water oxidation. The removal of Ca2+ rises the pK of B, thereby disturbing the hydrogen bonded structure of YZB
The molecular proceedings of biological hydrogen turnover
Over the past two decades, the bioinorganic chemistry of hydrogenases has attracted much interest from basic and applied research. Hydrogenases are highly efficient metalloenzymes that catalyze the reversible reduction of protons to molecular hydrogen (H2) in all domains of life. Their iron- and nickel-based cofactors represent promising blueprints for the design of biomimetic, synthetic catalysts. In this Account, we address the molecular proceedings of hydrogen turnover with [FeFe]-hydrogenases.
The active site cofactor of [FeFe]-hydrogenases (âH-clusterâ) comprises a unique diiron complex linked to a [4Fe-4S] cluster via a single cysteine. Since it was discovered that a synthetic analogue of the diiron site can be incorporated into apoprotein in vitro to yield active enzyme, significant progress has been made toward a comprehensive understanding of hydrogenase catalysis. The diiron site carries three to four carbon monoxide (CO) and two cyanide (CNâ) ligands that give rise to intense infrared (IR) absorption bands. These bands are sensitive reporters of the electron density across the H-cluster, which can be addressed by infrared spectroscopy to follow redox and protonation changes at the cofactor. Synthetic variation of the metal-bridging dithiolate ligand at the diiron site, as well as site-directed mutagenesis of amino acids, provides access to the proton pathways toward the cofactor. Quantum chemical calculations are employed to specifically assign IR bands to vibrational modes of the diatomic ligands and yield refined H-cluster geometries.
Here, we provide an overview of recent research on [FeFe]-hydrogenases with emphasis on experimental and computational IR studies. We describe advances in attenuated total reflection Fourier transform infrared spectroscopy (ATR FTIR) and protein film electrochemistry, as well as density functional theory (DFT) calculations. Key cofactor species are discussed in terms of molecular geometry, redox state, and protonation. Isotope editing is introduced as a tool to evaluate the cofactor geometry beyond the limits of protein crystallography. In particular, the role of proton-coupled electron transfer (PCET) in the generation of catalytically relevant redox species is addressed.
We propose that site-selective protonation of the H-cluster biases surplus electrons either to the [4Fe-4S] cluster or to the diiron site. Protonation of the [4Fe-4S] cluster prevents premature reduction at the diiron site and stabilizes a reactive, terminal hydride. The observed H-cluster species are assigned to rapid H2 conversion or to reactions possibly involved in activity regulation and cellular H2 sensing. In the catalytic cycle of [FeFe]-hydrogenases, an H-cluster geometry is preserved that features a bridging CO ligand. PCET levels the redox potential for two steps of sequential cofactor reduction. The concept of consecutive PCET at a geometrically inert cofactor with tight control of electron and proton localization may inspire the design of a novel generation of biomimetic catalysts for the production of H2 as a fuel
Alternating electron and proton transfer steps in photosynthetic water oxidation
Water oxidation by cyanobacteria, algae, and plants is pivotal in oxygenic
photosynthesis, the process that powers life on Earth, and is the paradigm for
engineering solar fuelâproduction systems. Each complete reaction cycle of
photosynthetic water oxidation requires the removal of four electrons and four
protons from the catalytic site, a manganeseâcalcium complex and its protein
environment in photosystem II. In time-resolved photothermal beam deflection
experiments, we monitored apparent volume changes of the photosystem II
protein associated with charge creation by light-induced electron transfer
(contraction) and charge-compensating proton relocation (expansion). Two
previously invisible proton removal steps were detected, thereby filling two
gaps in the basic reaction-cycle model of photosynthetic water oxidation. In
the S2 â S3 transition of the classical S-state cycle, an intermediate is
formed by deprotonation clearly before electron transfer to the oxidant
(Graphic). The rate-determining elementary step (Ï, approximately 30 ”s at 20
°C) in the long-distance proton relocation toward the proteinâwater interface
is characterized by a high activation energy (Ea = 0.46 ± 0.05 eV) and strong
H/D kinetic isotope effect (approximately 6). The characteristics of a proton
transfer step during the S0 â S1 transition are similar (Ï, approximately 100
”s; Ea = 0.34 ± 0.08 eV; kinetic isotope effect, approximately 3); however,
the proton removal from the Mn complex proceeds after electron transfer to
Graphic. By discovery of the transient formation of two further intermediate
states in the reaction cycle of photosynthetic water oxidation, a temporal
sequence of strictly alternating removal of electrons and protons from the
catalytic site is established
Photosynthetic oxygen evolution: Net charge transients as inferred from electrochromic bandshifts are independent of proton release into the medium
AbstractThe manganese containing center of the oxygen evolving complex accumulates four oxidizing equivalents in the four stepped water oxidation cycle. Based on experiments on electrochromic absorption transients and the reduction rate of the primary electron donor, P680, it has been speculated that the oscillations of these variables reflect the net charge of the center as calculated from the difference between electron abstraction and proton release into the medium. We compared proton release with electrochromism in thylakoids and core particles, and under variation of the rate of proton release. We found no equivalent of the variations of the extents and the rates of proton release in electrochromism. The oscillatory pattern of the latter reflects the topological properties of the stepped charge storage relative to the position and orientation of electrochromically responsive pigments rather than responding to proton release from the periphery
Identification of YdhV as the first molybdoenzyme binding a Bis-Mo-MPT cofactor in escherichia coli
The oxidoreductase YdhV in Escherichia coli has been predicted to belong to the family of molybdenum/tungsten cofactor (Moco/Wco)-containing enzymes. In this study, we characterized the YdhV protein in detail, which shares amino acid sequence homology with a tungsten-containing benzoyl-CoA reductase binding the bis-W-MPT (for metal-binding pterin) cofactor. The cofactor was identified to be of a bis-Mo-MPT type with no guanine nucleotides present, which represents a form of Moco that has not been found previously in any molybdoenzyme. Our studies showed that YdhV has a preference for bis-Mo-MPT over bis-W-MPT to be inserted into the enzyme. In-depth characterization of YdhV by X-ray absorption and electron paramagnetic resonance spectroscopies revealed that the bis-Mo-MPT cofactor in YdhV is redox active. The bis-Mo-MPT and bis-W-MPT cofactors include metal centers that bind the four sulfurs from the two dithiolene groups in addition to a cysteine and likely a sulfido ligand. The unexpected presence of a bis-Mo-MPT cofactor opens an additional route for cofactor biosynthesis in E. coli and expands the canon of the structurally highly versatile molybdenum and tungsten cofactors
Modelling the coordination environment in α-ketoglutarate dependent oxygenases â a comparative study on the effect of N- vs. O-ligation
In various non-heme iron oxygenases the Fe(II) center is coordinated by 2â
N and 1â
O atoms of the 2-His-2-carboxylate facial triad; however, most artificial model complexes bear only N-based ligands. In an effort to closely mimic the coordination environment in α-ketoglutarate dependent oxygenases, we have now employed the Me2tacnO ligand (4,7-dimethyl-1-oxa-4,7-diazacyclononane) in the synthesis of the complexes [(Me2tacnO)FeCl2]2 (1-NNO), [(Me2tacnO)FeCl3] (1âb-NNO) and [(Me2tacnO)Fe(BF)Cl] (2-NNO; BF=benzoylformate). The weaker donation of the O atom in the ligand was found to result in stronger binding of the ligand in trans-position to the O-atom of the ancillary ligand as compared to the corresponding complexes involving the Me3tacn (1,4,7-trimethyl-1,4,7-triazacyclononane) ligand. Furthermore, by stopped-flow techniques we could detect an intermediate (3-NNO) in the reaction of 2-NNO with O2. The spectroscopic features of 3-NNO agree with the involvement of an Fe(IV)-oxo intermediate and hence this study represents the first detection of such an intermediate in the O2 activation of artificial α-ketoglutarate Fe(II) complexes
Modelling the coordination environment in αâketoglutarate dependent oxygenases â a comparative study on the effect of Nâ vs. Oâligation
In various non-heme iron oxygenases the Fe(II) center is coordinated by 2â
N and 1â
O atoms of the 2-His-2-carboxylate facial triad; however, most artificial model complexes bear only N-based ligands. In an effort to closely mimic the coordination environment in α-ketoglutarate dependent oxygenases, we have now employed the Me2tacnO ligand (4,7-dimethyl-1-oxa-4,7-diazacyclononane) in the synthesis of the complexes [(Me2tacnO)FeCl2]2 (1-NNO), [(Me2tacnO)FeCl3] (1âb-NNO) and [(Me2tacnO)Fe(BF)Cl] (2-NNO; BF=benzoylformate). The weaker donation of the O atom in the ligand was found to result in stronger binding of the ligand in trans-position to the O-atom of the ancillary ligand as compared to the corresponding complexes involving the Me3tacn (1,4,7-trimethyl-1,4,7-triazacyclononane) ligand. Furthermore, by stopped-flow techniques we could detect an intermediate (3-NNO) in the reaction of 2-NNO with O2. The spectroscopic features of 3-NNO agree with the involvement of an Fe(IV)-oxo intermediate and hence this study represents the first detection of such an intermediate in the O2 activation of artificial α-ketoglutarate Fe(II) complexes.Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)Peer Reviewe
Comparison of geodetic and glaciological mass budgets for White Glacier, Axel Heiberg Island, Canada
Lyophilization protects [FeFe]-hydrogenases against O2-induced H-cluster degradation
Nature has developed an impressive repertoire of metal-based enzymes that
perform complex chemical reactions under moderate conditions. Catalysts that
produce molecular hydrogen (H2) are particularly promising for renewable
energy applications. Unfortunately, natural and chemical H2-catalysts are
often irreversibly degraded by molecular oxygen (O2). Here we present a
straightforward procedure based on freeze-drying (lyophilization), that turns
[FeFe]-hydrogenases, which are excellent H2-producers, but typically extremely
O2-sensitive in solution, into enzymes that are fully resistant against O2.
Complete dryness protects and conserves both, the [FeFe]-hydrogenase proteins
and their inorganic active-site cofactor (H-cluster), when exposed to 100% O2
for days. The full H2-formation capacity is restored after solvation of the
lyophilized enzymes. However, even minimal moisturizing re-establishes
O2-sensitivity. The dry [FeFe]-hydrogenase material is superior also for
advanced spectroscopic investigations on the H-cluster reaction mechanism. Our
method provides a convenient way for long-term storage and impacts on
potential biotechnological hydrogen production applications of hydrogenase
enzymes
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