398 research outputs found
Dynamic water bridging and proton transfer at a surface carboxylate cluster of photosystem II
Proton-transfer proteins are often exposed to the bulk clusters of carboxylate groups that might bind protons transiently. This raises important questions as to how the carboxylate groups of a protonated cluster interact with each other and with water, and how charged protein groups and hydrogen-bonded waters could have an impact on proton transfers at the cluster. We address these questions by combining classical mechanical and quantum mechanical computations with the analysis of cyanobacterial photosystem II crystal structures from Thermosynechococcus elongatus. The model system we use consists of an interface between PsbO and PsbU, which are two extrinsic proteins of photosystem II. We find that a protonated carboxylate pair of PsbO is part of a dynamic network of protein–water hydrogen bonds which extends across the protein interface. Hydrogen-bonded waters and a conserved lysine sidechain largely shape the energetics of proton transfer at the carboxylate cluster
Protein dynamics in the reductive activation of a B12-containing enzyme
B12-dependent proteins are involved in methyl transfer reactions ranging from the biosynthesis of methionine in humans to the formation of acetyl-CoA in anaerobic bacteria. During their catalytic cycle, they undergo large conformational changes to interact with various proteins. Recently, the crystal structure of the B12-containing corrinoid iron–sulfur protein (CoFeSP) in complex with its reductive activator (RACo) was determined, providing a first glimpse of how energy is transduced in the ATP-dependent reductive activation of corrinoid-containing methyltransferases. The thermodynamically uphill electron transfer from RACo to CoFeSP is accompanied by large movements of the cofactor-binding domains of CoFeSP. To refine the structure-based mechanism, we analyzed the conformational change of the B12-binding domain of CoFeSP by pulsed electron–electron double resonance and Förster resonance energy transfer spectroscopy. We show that the site-specific labels on the flexible B12-binding domain and the small subunit of CoFeSP move within 11 Å in the RACo:CoFeSP complex, consistent with the recent crystal structures. By analyzing the transient kinetics of formation and dissociation of the RACo:CoFeSP complex, we determined values of 0.75 μM–1 s–1 and 0.33 s–1 for rate constants kon and koff, respectively. Our results indicate that the large movement observed in crystals also occurs in solution and that neither the formation of the protein encounter complex nor the large movement of the B12-binding domain is rate-limiting for the ATP-dependent reductive activation of CoFeSP by RACo
Assignment of Individual Metal Redox States in a Metalloprotein by Crystallographic Refinement at Multiple X-ray Wavelengths
A method is presented to derive anomalous scattering contributions for individual atoms within a protein crystal by collecting several sets of diffraction data at energies spread along an X-ray absorption edge of the element in question. The method has been applied to a [2Fe:2S] ferredoxin model system with localized charges in the reduced state of the iron−sulfur cluster. The analysis shows that upon reduction the electron resides at the iron atom closer to the protein surface. The technique should be sufficiently sensitive for more complex clusters with noninteger redox states and is generally applicable given that crystals are available
Axial Ligation and Redox Changes at the Cobalt Ion in Cobalamin Bound to Corrinoid Iron-Sulfur Protein (CoFeSP) or in Solution Characterized by XAS and DFT
A cobalamin (Cbl) cofactor in corrinoid iron-sulfur protein (CoFeSP) is the
primary methyl group donor and acceptor in biological carbon oxide conversion
along the reductive acetyl-CoA pathway. Changes of the axial coordination of
the cobalt ion within the corrin macrocycle upon redox transitions in aqua-,
methyl-, and cyano-Cbl bound to CoFeSP or in solution were studied using X-ray
absorption spectroscopy (XAS) at the Co K-edge in combination with density
functional theory (DFT) calculations, supported by metal content and cobalt
redox level quantification with further spectroscopic methods. Calculation of
the highly variable pre-edge X-ray absorption features due to core-to-valence
(ctv) electronic transitions, XANES shape analysis, and cobalt-ligand bond
lengths determination from EXAFS has yielded models for the molecular and
electronic structures of the cobalt sites. This suggested the absence of a
ligand at cobalt in CoFeSP in α-position where the dimethylbenzimidazole (dmb)
base of the cofactor is bound in Cbl in solution. As main species,
(dmb)CoIII(OH2), (dmb)CoII(OH2), and (dmb)CoIII(CH3) sites for solution Cbl
and CoIII(OH2), CoII(OH2), and CoIII(CH3) sites in CoFeSP-Cbl were identified.
Our data support binding of a serine residue from the reductive-activator
protein (RACo) of CoFeSP to the cobalt ion in the CoFeSP-RACo protein complex
that stabilizes Co(II). The absence of an α-ligand at cobalt not only tunes
the redox potential of the cobalamin cofactor into the physiological range,
but is also important for CoFeSP reactivation
a FD-FT THz-EPR study
A combined X-band and frequency-domain Fourier-transform THz electron
paramagnetic resonance (FD-FT THz-EPR) approach has been employed to determine
heme Fe(III) S = 5/2 zero-field splitting (ZFS) parameters of frozen metHb and
metMb solutions, both with fluoro and aquo ligands. Frequency-domain EPR
measurements have been carried out by an improved synchrotron-based FD-FT THz-
EPR spectrometer. ZFS has been determined by field dependence of spin
transitions within the mS = ±1/2 manifold, for all four protein systems, and
by zero-field spin transitions between mS = ±1/2 and mS = ±3/2 levels, for
metHb and metMb flouro-states. FD-FT THz-EPR data were simulated with a novel
numerical routine based on Easyspin, which allows now for direct comparison of
EPR spectra in field and frequency domain. We found purely axial ZFSs of D =
5.0(1) cm−1 (flouro-metMb), D = 9.2(4) cm−1 (aquo-metMb), D = 5.1(1) cm−1
(flouro-metHB) and D = 10.4(2) cm−1 (aquo-metHb)
A Morphing [4Fe-3S-nO]-Cluster within a Carbon Monoxide Dehydrogenase Scaffold
Ni,Fe-containing carbon monoxide dehydrogenases (CODHs) catalyze the reversible reduction of CO2 to CO. Several anaerobic microorganisms encode multiple CODHs in their genome, of which some, despite being annotated as CODHs, lack a cysteine of the canonical binding motif for the active site Ni,Fe-cluster. Here, we report on the structure and reactivity of such a deviant enzyme, termed CooS-VCh. Its structure reveals the typical CODH scaffold, but contains an iron-sulfur-oxo hybrid-cluster. Although closely related to true CODHs, CooS-VCh catalyzes neither CO oxidation, nor CO2 reduction. The active site of CooS-VCh undergoes a redox-dependent restructuring between a reduced [4Fe-3S]-cluster and an oxidized [4Fe-2S-S*-2O-2(H2O)]-cluster. Hydroxylamine, a slow-turnover substrate of CooS-VCh, oxidizes the hybrid-cluster in two structurally distinct steps. Overall, minor changes in CODHs are sufficient to accommodate a Fe/S/O-cluster in place of the Ni,Fe-heterocubane-cluster of CODHs
Structural insights into the light-driven auto-assembly process of the water- oxidizing Mn4CaO5-cluster in photosystem II
In plants, algae and cyanobacteria, Photosystem II (PSII) catalyzes the light-
driven splitting of water at a protein-bound Mn4CaO5-cluster, the water-
oxidizing complex (WOC). In the photosynthetic organisms, the light-driven
formation of the WOC from dissolved metal ions is a key process because it is
essential in both initial activation and continuous repair of PSII. Structural
information is required for understanding of this chaperone-free metal-cluster
assembly. For the first time, we obtained a structure of PSII from
Thermosynechococcus elongatus without the Mn4CaO5-cluster. Surprisingly,
cluster-removal leaves the positions of all coordinating amino acid residues
and most nearby water molecules largely unaffected, resulting in a pre-
organized ligand shell for kinetically competent and error-free photo-assembly
of the Mn4CaO5-cluster. First experiments initiating (i) partial disassembly
and (ii) partial re-assembly after complete depletion of the Mn4CaO5-cluster
agree with a specific bi-manganese cluster, likely a di-µ-oxo bridged pair of
Mn(III) ions, as an assembly intermediate
A role for nickel-iron cofactors in biological carbon monoxide and carbon dioxide utilization
Ni–Fe containing enzymes are involved in the biological utilization of carbon monoxide, carbon dioxide, and hydrogen. Interest in these enzymes has increased in recent years due to hydrogen fuel initiatives and concerns over development of new methods for CO2 sequestration. One Ni–Fe enzyme called carbon monoxide dehydrogenase (CODH) is a key player in the global carbon cycle and carries out the interconversion of the environmental pollutant CO and the greenhouse gas CO[subscript 2]. The Ni–Fe center responsible for this important chemistry, the C-cluster, has been the source of much controversy, but several recent structural studies have helped to direct the field toward a unifying mechanism. Here we summarize the current state of understanding of this fascinating metallocluster.National Institutes of Health (U.S.) (GM69857)Massachusetts Institute of Technology. Energy InitiativeHoward Hughes Medical Institute. Investigato
Determining the oxidation state of elements by X ray crystallography
Protein-mediated redox reactions play a critical role in many biological processes and often occur at centres that contain metal ions as cofactors. In order to understand the exact mechanisms behind these reactions it is important to not only characterize the three-dimensional structures of these proteins and their cofactors, but also to identify the oxidation states of the cofactors involved and to correlate this knowledge with structural information. The only suitable approach for this based on crystallographic measurements is spatially resolved anomalous dispersion (SpReAD) refinement, a method that has been used previously to determine the redox states of metals in iron–sulfur cluster-containing proteins. In this article, the feasibility of this approach for small, non-iron–sulfur redox centres is demonstrated by employing SpReAD analysis to characterize Sulfolobus tokodaii sulerythrin, a ruberythrin-like protein that contains a binuclear metal centre. Differences in oxidation states between the individual iron ions of the binuclear metal centre are revealed in sulerythrin crystals treated with H(2)O(2). Furthermore, data collection at high X-ray doses leads to photoreduction of this metal centre, showing that careful control of the total absorbed dose is a prerequisite for successfully determining the oxidation state through SpReAD analysis
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