RESUMEN
Charge exchange is the fundamental process that sustains cellular respiration and photosynthesis by shuttling electrons in a cascade of electron transfer (ET) steps between redox cofactors. While intraprotein charge exchange is well characterized in protein complexes bearing multiple redox sites, interprotein processes are less understood due to the lack of suitable experimental approaches and the dynamic nature of the interactions. Proteins constrained between electrodes are known to support electron transport (ETp) through the protein matrix even without redox cofactors, as the charges housed by the redox sites in ET are furnished by the electrodes. However, it is unknown whether protein ETp mechanisms apply to the interprotein medium present under physiological conditions. We study interprotein charge exchange between plant photosystem I (PSI) and its soluble redox partner plastocyanin (Pc) and address the role of the Pc copper center. Using electrochemical scanning tunneling spectroscopy (ECSTS) current-distance and blinking measurements, we quantify the spatial span of charge exchange between individual Pc/PSI pairs and ETp through transient Pc/PSI complexes. Pc devoid of the redox center (Pcapo) can exchange charge with PSI at longer distances than with the copper ion (Pcholo). Conductance bursts associated with Pcapo/PSI complex formation are higher than in Pcholo/PSI. Thus, copper ions are not required for long-distance Pc/PSI ETp but regulate its spatial span and conductance. Our results suggest that the redox center that carries the charge in Pc is not necessary to exchange it in interprotein ET through the aqueous solution and question the canonical view of tight complex binding between redox protein partners.
Asunto(s)
Complejo de Proteína del Fotosistema I , Plastocianina , Complejo de Proteína del Fotosistema I/química , Complejo de Proteína del Fotosistema I/metabolismo , Plastocianina/química , Plastocianina/metabolismo , Cobre , Transporte de Electrón , Oxidación-Reducción , Plantas/metabolismoRESUMEN
Proteins tune the reactivity of metal sites; less understood is the impact of association with a redox partner. We demonstrate the utility of carbon-deuterium labels for selective analysis of delicate metal sites. Introduced into plastocyanin, they reveal substantial strengthening of the key Cu-Cys89 bond upon association with cytochrome f.
Asunto(s)
Cobre , Plastocianina , Carbono , Cobre/química , Citocromos f/metabolismo , Deuterio , Oxidación-Reducción , Plastocianina/química , Plastocianina/metabolismoRESUMEN
Photosynthesis is a fundamental process that converts photons into chemical energy, driven by large protein complexes at the thylakoid membranes of plants, cyanobacteria, and algae. In plants, water-soluble plastocyanin (Pc) is responsible for shuttling electrons between cytochrome b6f complex and the photosystem I (PSI) complex in the photosynthetic electron transport chain (PETC). For an efficient turnover, a transient complex must form between PSI and Pc in the PETC, which implies a balance between specificity and binding strength. Here, we studied the binding frequency and the unbinding force between suitably oriented plant PSI and Pc under redox control using single molecule force spectroscopy (SMFS). The binding frequency (observation of binding-unbinding events) between PSI and Pc depends on their respective redox states. The interaction between PSI and Pc is independent of the redox state of PSI when Pc is reduced, and it is disfavored in the dark (reduced P700) when Pc is oxidized. The frequency of interaction between PSI and Pc is higher when at least one of the partners is in a redox state ready for electron transfer (ET), and the post-ET situation (PSIRed-PcOx) leads to lower binding. In addition, we show that the binding of ET-ready PcRed to PSI can be regulated externally by Mg2+ ions in solution.
Asunto(s)
Complejo de Proteína del Fotosistema I , Plastocianina , Complejo de Citocromo b6f/química , Complejo de Citocromo b6f/metabolismo , Transporte de Electrón , Electrones , Luz , Oxidación-Reducción , Complejo de Proteína del Fotosistema I/química , Complejo de Proteína del Fotosistema I/metabolismo , Plastocianina/química , Plastocianina/metabolismo , Análisis Espectral , Agua/metabolismoRESUMEN
In the light reaction of oxygenic photosynthesis, plastocyanin (PC) and ferredoxins (Fd) are small/diffusible redox-active proteins playing key roles in electron transfer/transport phenomena. In the Z-scheme mechanistic purview, they are considered as specific affinity binding-based electron-relay agents, linking the functions of Cytochrome b6f (Cyt. b6f), Photosystem I (PS I) and Fd:NADPH oxidoreductase (FNR). The murburn explanation for photolytic photophosphorylation deems PC/Fd as generic 'redox capacitors', temporally accepting and releasing one-electron equivalents in reaction milieu. Herein, we explore the two theories with respect to structural, distributional and functional aspects of PC/Fd. Amino acid residues located on the surface loci of key patches of PC/Fd vary in electrostatic/contour (topography) signatures. Crystal structures of four different complexes each of Cyt.f-PC and Fd-FNR show little conservation in the contact-surfaces, thereby discrediting 'affinity binding-based electron transfers (ET)' as an evolutionary logic. Further, thermodynamic and kinetic data of wildtype and mutant proteins interactions do not align with Z-scheme. Furthermore, micromolar physiological concentrations of PC and the non-conducive architecture of chloroplasts render the classical model untenable. In the murburn model, as PC is optional, the observation that plants lacking PC survive and grow is justified. Further, the low physiological concentration/distribution of PC in chloroplast lumen/stroma is supported by murburn equilibriums, as higher concentrations would limit electron transfers. Thus, structural evidence, interactive dynamics with redox partners and physiological distribution/role of PC/Fd support the murburn perspective that these proteins serve as generic redox-capacitors in chloroplasts.Communicated by Ramaswamy H. Sarma.
Asunto(s)
Ferredoxinas , Plastocianina , Transporte de Electrón , Electrones , Ferredoxinas/química , Ferredoxinas/metabolismo , Oxidación-Reducción , Fotosíntesis , Plastocianina/química , Plastocianina/metabolismoRESUMEN
Photosystem I is defined as plastocyanin-ferredoxin oxidoreductase. Taking advantage of genetic engineering, kinetic analyses and cryo-EM, our data provide novel mechanistic insights into binding and electron transfer between PSI and Pc. Structural data at 2.74â Å resolution reveals strong hydrophobic interactions in the plant PSI-Pc ternary complex, leading to exclusion of water molecules from PsaA-PsaB/Pc interface once the PSI-Pc complex forms. Upon oxidation of Pc, a slight tilt of bound oxidized Pc allows water molecules to accommodate the space between Pc and PSI to drive Pc dissociation. Such a scenario is consistent with the six times larger dissociation constant of oxidized as compared with reduced Pc and mechanistically explains how this molecular machine optimized electron transfer for fast turnover.
Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Complejo de Proteína del Fotosistema I/química , Complejo de Proteína del Fotosistema I/metabolismo , Plastocianina/química , Plastocianina/metabolismo , Sitios de Unión , Transporte de Electrón , Cinética , Modelos Moleculares , Oxidación-Reducción , Unión Proteica , Conformación ProteicaRESUMEN
Many cyanobacteria species can use both plastocyanin and cytochrome c6 as lumenal electron carriers to shuttle electrons from the cytochrome b6f to either photosystem I or the respiratory cytochrome c oxidase. In Synechocystis sp. PCC6803 placed in darkness, about 60% of the active PSI centres are bound to a reduced electron donor which is responsible for the fast re-reduction of P700in vivo after a single charge separation. Here, we show that both cytochrome c6 and plastocyanin can bind to PSI in the dark and participate to the fast phase of P700 reduction, but the fraction of pre-bound PSI is smaller in the case of cytochrome c6 than with plastocyanin. Because of the inter-connection of respiration and photosynthesis in cyanobacteria, the inhibition of the cytochrome c oxidase results in the over-reduction of the photosynthetic electron transfer chain in the dark that translates into a lag in the kinetics of P700 oxidation at the onset of light. We show that this is true both with plastocyanin and cytochrome c6, indicating that the partitioning of electron transport between respiration and photosynthesis is regulated in the same way independently of which of the two lumenal electron carriers is present, although the mechanisms of such regulation are yet to be understood.
Asunto(s)
Citocromos c6/química , Complejo de Proteína del Fotosistema I/química , Plastocianina/química , Synechocystis/metabolismo , Clorofila/química , Cianobacterias/metabolismo , Transporte de Electrón , Complejo IV de Transporte de Electrones/química , Cinética , Oxidación-Reducción , Fotosíntesis , Tilacoides/químicaRESUMEN
The ability of photosynthetic organisms to use sunlight as a sole source of energy is endowed by two large membrane complexes-photosystem I (PSI) and photosystem II (PSII). PSI and PSII are the fundamental components of oxygenic photosynthesis, providing oxygen, food and an energy source for most living organisms on Earth. Currently, high-resolution crystal structures of these complexes from various organisms are available. The crystal structures of megadalton complexes have revealed excitation transfer and electron-transport pathways within the various complexes. PSI is defined as plastocyanin-ferredoxin oxidoreductase but a high-resolution structure of the entire triple supercomplex is not available. Here, using a new cryo-electron microscopy technique, we solve the structure of native plant PSI in complex with its electron donor plastocyanin and the electron acceptor ferredoxin. We reveal all of the contact sites and the modes of interaction between the interacting electron carriers and PSI.
Asunto(s)
Ferredoxinas/ultraestructura , Complejo de Proteína del Fotosistema I/ultraestructura , Pisum sativum/ultraestructura , Plastocianina/ultraestructura , Sitios de Unión , Microscopía por Crioelectrón , Electrones , Ferredoxinas/química , Modelos Moleculares , Complejo de Proteína del Fotosistema I/química , Plastocianina/química , Conformación ProteicaRESUMEN
A kinetic-LED-array-spectrophotometer (Klas) was recently developed for measuring in vivo redox changes of P700, plastocyanin (PCy), and ferredoxin (Fd) in the near-infrared (NIR). This spectrophotometer is used in the present work for in vitro light-induced measurements with various combinations of photosystem I (PSI) from tobacco and two different cyanobacteria, spinach plastocyanin, cyanobacterial cytochrome c6 (cyt. c6), and Fd. It is shown that cyt. c6 oxidation contributes to the NIR absorption changes. The reduction of (FAFB), the terminal electron acceptor of PSI, was also observed and the shape of the (FAFB) NIR difference spectrum is similar to that of Fd. The NIR difference spectra of the electron-transfer cofactors were compared between different organisms and to those previously measured in vivo, whereas the relative absorption coefficients of all cofactors were determined by using single PSI turnover conditions. Thus, the (840 nm minus 965 nm) extinction coefficients of the light-induced species (oxidized minus reduced for PC and cyt. c6, reduced minus oxidized for (FAFB), and Fd) were determined with values of 0.207 ± 0.004, - 0.033 ± 0.006, - 0.036 ± 0.008, and - 0.021 ± 0.005 for PCy, cyt. c6, (FAFB) (single reduction), and Fd, respectively, by taking a reference value of + 1 for P700+. The fact that the NIR P700 coefficient is larger than that of PCy and much larger than that of other contributing species, combined with the observed variability in the NIR P700 spectral shape, emphasizes that deconvolution of NIR signals into different components requires a very precise determination of the P700 spectrum.
Asunto(s)
Proteínas Bacterianas/química , Complejo de Proteína del Fotosistema I/metabolismo , Proteínas de Plantas/química , Espectroscopía Infrarroja Corta/métodos , Proteínas Bacterianas/metabolismo , Citocromos c6/química , Citocromos c6/metabolismo , Transporte de Electrón , Ferredoxinas/metabolismo , Oxidación-Reducción , Complejo de Proteína del Fotosistema I/química , Proteínas de Plantas/metabolismo , Plastocianina/química , Plastocianina/metabolismo , Espectrofotometría Ultravioleta , Espectroscopía Infrarroja Corta/instrumentación , Spinacia oleracea/química , Synechocystis/química , Nicotiana/químicaRESUMEN
In plants and green algae, the core of photosystem I (PSI) is surrounded by a peripheral antenna system consisting of light-harvesting complex I (LHCI). Here we report the cryo-electron microscopic structure of the PSI-LHCI supercomplex from the green alga Chlamydomonas reinhardtii. The structure reveals that eight Lhca proteins form two tetrameric LHCI belts attached to the PsaF side while the other two Lhca proteins form an additional Lhca2/Lhca9 heterodimer attached to the opposite side. The spatial arrangement of light-harvesting pigments reveals that Chlorophylls b are more abundant in the outer LHCI belt than in the inner LHCI belt and are absent from the core, thereby providing the downhill energy transfer pathways to the PSI core. PSI-LHCI is complexed with a plastocyanin on the patch of lysine residues of PsaF at the luminal side. The assembly provides a structural basis for understanding the mechanism of light-harvesting, excitation energy transfer of the PSI-LHCI supercomplex and electron transfer with plastocyanin.
Asunto(s)
Chlamydomonas reinhardtii/ultraestructura , Complejos de Proteína Captadores de Luz/ultraestructura , Complejo de Proteína del Fotosistema I/química , Complejo de Proteína del Fotosistema I/ultraestructura , Transferencia de Energía , Proteínas de la Membrana/química , Modelos Moleculares , Plastocianina/química , Conformación Proteica , Especificidad de la EspecieRESUMEN
Small diffusible redox proteins play a ubiquitous role in bioenergetic systems, facilitating electron transfer (ET) between membrane bound complexes. Sustaining high ET turnover rates requires that the association between extrinsic and membrane-bound partners is highly specific, yet also sufficiently weak to promote rapid post-ET separation. In oxygenic photosynthesis the small soluble electron carrier protein plastocyanin (Pc) shuttles electrons between the membrane integral cytochrome b6f (cytb6f) and photosystem I (PSI) complexes. Here we use peak-force quantitative nanomechanical mapping (PF-QNM) atomic force microscopy (AFM) to quantify the dynamic forces involved in transient interactions between cognate ET partners. An AFM probe functionalised with Pc molecules is brought into contact with cytb6f complexes, immobilised on a planar silicon surface. PF-QNM interrogates the unbinding force of the cytb6f-Pc interactions at the single molecule level with picoNewton force resolution and on a time scale comparable to the ET time in vivo (ca. 120⯵s). Using this approach, we show that although the unbinding force remains unchanged the interaction frequency increases over five-fold when Pc and cytb6f are in opposite redox states, so complementary charges on the cytb6f and Pc cofactors likely contribute to the electrostatic forces that initiate formation of the ET complex. These results suggest that formation of the docking interface is under redox state control, which lowers the probability of unproductive encounters between Pc and cytb6f molecules in the same redox state, ensuring the efficiency and directionality of this central reaction in the 'Z-scheme' of photosynthetic ET.
Asunto(s)
Complejo de Citocromo b6f/metabolismo , Plastocianina/metabolismo , Análisis de la Célula Individual/métodos , Spinacia oleracea/metabolismo , Complejo de Citocromo b6f/química , Transporte de Electrón , Oxidación-Reducción , Fotosíntesis , Plastocianina/química , Unión Proteica , Conformación ProteicaRESUMEN
Alignment of molecules through electric fields minimizes the averaging over orientations, e.g., in single-particle-imaging experiments. The response of molecules to external ac electric fields is governed by their polarizability tensor, which is usually calculated using quantum chemistry methods. These methods are not feasible for large molecules. Here, we calculate the polarizability tensor of proteins using a regression model that correlates the polarizabilities of the 20 amino acids with perfect conductors of the same shape. The dielectric constant of the molecules could be estimated from the slope of the regression line based on the Clausius-Mossotti equation. We benchmark our predictions against the quantum chemistry results for the Trp cagemini protein and the measured dielectric constants of larger proteins. Our method has applications in computing laser alignment of macromolecules, for instance, benefiting single-particle imaging, as well as for estimation of the optical and electrostatic characteristics of proteins and other macromolecules.
Asunto(s)
Aminoácidos/química , Simulación por Computador , Anabaena variabilis/química , Cianobacterias/química , Inhibidor de la Unión a Diazepam/química , Glutarredoxinas/química , Humanos , Plastocianina/química , Teoría Cuántica , Análisis de Regresión , Electricidad EstáticaRESUMEN
Proteins exist as ensembles of interconverting states on a complex energy landscape. A complete, molecular-level understanding of their function requires knowledge of the populated states and thus the experimental tools to characterize them. Infrared (IR) spectroscopy has an inherently fast time scale that can capture all states and their dynamics with, in principle, bond-specific spatial resolution, and 2D IR methods that provide richer information are becoming more routine. Although application of IR spectroscopy for investigation of proteins is challenged by spectral congestion, the issue can be overcome by site-specific introduction of amino acid side chains that have IR probe groups with frequency-resolved absorptions, which furthermore enables selective characterization of different locations in proteins. Here, we briefly introduce the biophysical methods and summarize the current progress toward the study of proteins. We then describe our efforts to apply site-specific 1D and 2D IR spectroscopy toward elucidation of protein conformations and dynamics to investigate their involvement in protein molecular recognition, in particular mediated by dynamic complexes: plastocyanin and its binding partner cytochrome f, cytochrome P450s and substrates or redox partners, and Src homology 3 domains and proline-rich peptide motifs. We highlight the advantages of frequency-resolved probes to characterize specific, local sites in proteins and uncover variation among different locations, as well as the advantage of the fast time scale of IR spectroscopy to detect rapidly interconverting states. In addition, we illustrate the greater insight provided by 2D methods and discuss potential routes for further advancement of the field of biomolecular 2D IR spectroscopy.
Asunto(s)
Sistema Enzimático del Citocromo P-450/química , Citocromos f/química , Simulación de Dinámica Molecular , Plastocianina/química , Modelos Moleculares , Conformación Proteica , Espectrofotometría InfrarrojaRESUMEN
Transient protein complexes are crucial for sustaining dynamic cellular processes. The complexes of electron-transfer proteins are a notable example, such as those formed by plastocyanin (Pc) and cytochrome f (cyt f) in the photosynthetic apparatus. The dynamic and heterogeneous nature of these complexes, however, makes their study challenging. To better elucidate the complex of Nostoc Pc and cyt f, 2D-IR spectroscopy coupled to site-specific labeling with cyanophenylalanine infrared (IR) probes was employed to characterize how the local environments at sites along the surface of Pc were impacted by cyt f binding. The results indicate that Pc most substantially engages with cyt f via the hydrophobic patch around the copper redox site. Complexation with cyt f led to an increase in inhomogeneous broadening of the probe absorptions, reflective of increased heterogeneity of interactions with their environment. Notably, most of the underlying states interconverted very rapidly (1 to 2 ps), suggesting a complex with a highly mobile interface. The data support a model of the complex consisting of a large population of an encounter complex. Additionally, the study demonstrates the application of 2D-IR spectroscopy with site-specifically introduced probes to reveal new quantitative insight about dynamic biochemical systems.
Asunto(s)
Citocromos f/metabolismo , Plastocianina/metabolismo , Alanina/análogos & derivados , Alanina/química , Sitios de Unión , Citocromos f/química , Interacciones Hidrofóbicas e Hidrofílicas , Sondas Moleculares/química , Nitrilos/química , Nostoc/química , Plastocianina/química , Unión Proteica , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
We investigate the effects of solvent specificities on the stability of the native structure (NS) of a protein on the basis of our free-energy function (FEF). We use CPB-bromodomain (CBP-BD) and apoplastocyanin (apoPC) as representatives of the protein universe and water, methanol, ethanol, and cyclohexane as solvents. The NSs of CBP-BD and apoPC consist of 66% α-helices and of 35% ß-sheets and 4% α-helices, respectively. In order to assess the structural stability of a given protein immersed in each solvent, we contrast the FEF of its NS against that of a number of artificially created, misfolded decoys possessing the same amino-acid sequence but significantly different topology and α-helix and ß-sheet contents. In the FEF, we compute the solvation entropy using the morphometric approach combined with the integral equation theories, and the change in electrostatic (ES) energy upon the folding is obtained by an explicit atomistic but simplified calculation. The ES energy change is represented by the break of protein-solvent hydrogen bonds (HBs), formation of protein intramolecular HBs, and recovery of solvent-solvent HBs. Protein-solvent and solvent-solvent HBs are absent in cyclohexane. We are thus able to separately evaluate the contributions to the structural stability from the entropic and energetic components. We find that for both CBP-BD and apoPC, the energetic component dominates in methanol, ethanol, and cyclohexane, with the most stable structures in these solvents sharing the same characteristics described as an association of α-helices. In particular, those in the two alcohols are identical. In water, the entropic component is as strong as or even stronger than the energetic one, with a large gain of translational, configurational entropy of water becoming crucially important so that the relative contents of α-helix and ß-sheet and the content of total secondary structures are carefully selected to achieve sufficiently close packing of side chains. If the energetic component is excluded for a protein in water, the priority is given to closest side-chain packing, giving rise to the formation of a structure with very low α-helix and ß-sheet contents. Our analysis, which requires minimal computational effort, can be applied to any protein immersed in any solvent and provides robust predictions that are quite consistent with the experimental observations for proteins in different solvent environments, thus paving the way toward a more detailed understanding of the folding process.
Asunto(s)
Apoproteínas/química , Plastocianina/química , Solventes/química , Enlace de Hidrógeno , Conformación Proteica , Dominios Proteicos , Electricidad EstáticaRESUMEN
Increasing the thermal stability of immobilized proteins is a motivating goal for improving the performance of electrochemical biodevices. In this work, we propose the immobilization of crosslinked plastocyanin from the thermophilic cyanobacterium Phormidium laminosum by simultaneous incubation of a mixture of plastocyanin and the coupling reagents. The thermal stability of the so built covalently immobilized protein films has been assessed by cyclic voltammetry in the 0-90⯰C temperature range and has been compared to that of physisorbed films. It is shown that the protein loss along a thermal cycle is significantly reduced in the case of the crosslinked films, whose redox properties remain unaltered along a cyclic heating-cooling thermal scan, and can withstand the contact with 70⯰C solutions for four hours. Comparison of thermal unfolding curves obtained by circular dichroism spectroscopy of both free and crosslinked protein confirms the improved thermic resistance of the crosslinked plastocyanin. Notably, the electron transfer thermodynamics of physisorbed and crosslinked plastocyanin films are quite similar, suggesting that the formation of intra- and inter-protein amide bonds do not affect the integrity and functionality of the copper redox centers. UV-Vis absorption and circular dichroism measurements corroborate that protein crosslinking does not alter the coordination geometry of the metal center.
Asunto(s)
Cianobacterias/química , Electrodos , Oro/química , Plastocianina/química , Proteínas/química , Dicroismo Circular , Cobre/química , Reactivos de Enlaces Cruzados/química , Calor , Oxidación-Reducción , Estabilidad Proteica , Espectrofotometría Ultravioleta , TermodinámicaRESUMEN
Oxidoreductases catalyze electron transfer reactions that ultimately provide the energy for life. A limited set of ancestral protein-metal modules are presumably the building blocks that evolved into this diverse protein family. However, the identity of these modules and their path to modern oxidoreductases is unknown. Using a comparative structural analysis approach, we identify a set of fundamental electron transfer modules that have evolved to form the extant oxidoreductases. Using transition metal-containing cofactors as fiducial markers, it is possible to cluster cofactor microenvironments into as few as four major modules: bacterial ferredoxin, cytochrome c, symerythrin, and plastocyanin-type folds. From structural alignments, it is challenging to ascertain whether modules evolved from a single common ancestor (homology) or arose by independent convergence on a limited set of structural forms (analogy). Additional insight into common origins is contained in the spatial adjacency network (SPAN), which is based on proximity of modules in oxidoreductases containing multiple cofactor electron transfer chains. Electron transfer chains within complex modern oxidoreductases likely evolved through repeated duplication and diversification of ancient modular units that arose in the Archean eon.
Asunto(s)
Coenzimas/metabolismo , Evolución Molecular , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Coenzimas/química , Citocromos c/química , Citocromos c/metabolismo , Transporte de Electrón , Ferredoxinas/química , Ferredoxinas/metabolismo , Metales/química , Metales/metabolismo , Modelos Moleculares , Plastocianina/química , Plastocianina/metabolismo , Conformación Proteica , Homología Estructural de ProteínaRESUMEN
Plastocyanin is a copper containing protein that is involved in the electron transfer process in photosynthetic organisms. The active site of plastocyanin is described as an entatic state whereby its structure represents a compromise between the structures favored by the oxidized and reduced forms. In this study, the nature of the entatic state is investigated through density functional theory-based hybrid quantum mechanics/molecular mechanics (QM/MM) molecular dynamics simulations. The strain energy is computed to be 12.8 kcal/mol and 14.5 kcal/mol for the oxidized and reduced forms of the protein, indicating that the active site has an intermediate structure. It is shown that the energy gap between the oxidized and reduced forms varies significantly with the fluctuations in the structure of the active site at room temperature. An accurate determination of the reorganization energy requires averaging over conformation and a large region of the protein around the active site to be treated at the quantum mechanical level. © 2016 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc.
Asunto(s)
Simulación de Dinámica Molecular , Plastocianina/química , Dominio Catalítico , Transporte de Electrón , Oxidación-Reducción , Conformación Proteica , Teoría Cuántica , TermodinámicaRESUMEN
Electrostatic interaction of plastocyanin and cytochrome f in the process of protein-protein complex formation was investigated by computer simulation methods. It was shown that long-range electrostatic interaction promotes energetically favorable mutual orientation of protein molecules at distances between their cofactors shorter than 5 nm. At distances shorter than 3 nm, these electrostatic interactions lead to a significantly detectable increase in the rate of convergence of the cofactors.
Asunto(s)
Citocromos f/química , Difusión , Proteínas de Plantas/química , Plastocianina/química , Electricidad Estática , Brassica napus , Simulación por Computador , Cobre/química , Modelos Químicos , Oxidación-Reducción , Programas Informáticos , Soluciones , Solventes/química , Spinacia oleraceaRESUMEN
The reactivity of metal sites in proteins is tuned by protein-based ligands. For example, in blue copper proteins such as plastocyanin (Pc), the structure imparts a highly elongated bond between the Cu and a methionine (Met) axial ligand to modulate its redox properties. Despite extensive study, a complete understanding of the contribution of the protein to redox activity is challenged by experimentally accessing both redox states of metalloproteins. Using infrared (IR) spectroscopy in combination with site-selective labeling with carbon-deuterium (C-D) vibrational probes, we characterized the localized changes at the Cu ligand Met97 in the oxidized and reduced states, as well as the Zn(II) or Co(II)-substituted, the pH-induced low-coordinate, the apoprotein, and the unfolded states. The IR absorptions of (d3-methyl)Met97 are highly sensitive to interaction of the sulfur-based orbitals with the metal center and are demonstrated to be useful reporters of its modulation in the different states. Unrestricted Kohn-Sham density functional theory calculations performed on a model of the Cu site of Pc confirm the observed dependence. IR spectroscopy was then applied to characterize the impact of binding to the physiological redox partner cytochrome (cyt) f. The spectral changes suggest a slightly stronger Cu-S(Met97) interaction in the complex with cyt f that has potential to modulate the electron transfer properties. Besides providing direct, molecular-level comparison of the oxidized and reduced states of Pc from the perspective of the axial Met ligand and evidence for perturbation of the Cu site properties by redox partner binding, this study demonstrates the localized spatial information afforded by IR spectroscopy of selectively incorporated C-D probes.
Asunto(s)
Cobre/química , Metionina/química , Plastocianina/química , Sitios de Unión , Ligandos , Metionina/genética , Modelos Moleculares , Nostoc/química , Oxidación-Reducción , Plastocianina/genética , Unión Proteica , Espectrofotometría Infrarroja , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
In this work, we characterized the intermolecular electron transfer (ET) properties of a de novo designed metallopeptide using laser-flash photolysis. α3D-CH3 is three helix bundle peptide that was designed to contain a copper ET site that is found in the ß-barrel fold of native cupredoxins. The ET activity of Cuα3D-CH3 was determined using five different photosensitizers. By exhibiting a complete depletion of the photo-oxidant and the successive formation of a Cu(II) species at 400 nm, the transient and generated spectra demonstrated an ET transfer reaction between the photo-oxidant and Cu(I)α3D-CH3. This observation illustrated our success in integrating an ET center within a de novo designed scaffold. From the kinetic traces at 400 nm, first-order and bimolecular rate constants of 10(5) s(-1) and 10(8) M(-1) s(-1) were derived. Moreover, a Marcus equation analysis on the rate versus driving force study produced a reorganization energy of 1.1 eV, demonstrating that the helical fold of α3D requires further structural optimization to efficiently perform ET. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.