Conjuring up a ghost: structural and functional characterization of FhuF, a ferric siderophore reductase from E. coli.

Iron is a fundamental element for virtually all forms of life. Despite its abundance, its bioavailability is limited, and thus, microbes developed siderophores, small molecules, which are synthesized inside the cell and then released outside for iron scavenging. Once inside the cell, iron removal does not occur spontaneously, instead this process is mediated by siderophore-interacting proteins (SIP) and/or by ferric-siderophore reductases (FSR). In the past two decades, representatives of the SIP subfamily have been structurally and biochemically characterized; however, the same was not achieved for the FSR subfamily. Here, we initiate the structural and functional characterization of FhuF, the first and only FSR ever isolated. FhuF is a globular monomeric protein mainly composed by α-helices sheltering internal cavities in a fold resembling the "palm" domain found in siderophore biosynthetic enzymes. Paramagnetic NMR spectroscopy revealed that the core of the cluster has electronic properties in line with those of previously characterized 2Fe-2S ferredoxins and differences appear to be confined to the coordination of Fe(III) in the reduced protein. In particular, the two cysteines coordinating this iron appear to have substantially different bond strengths. In similarity with the proteins from the SIP subfamily, FhuF binds both the iron-loaded and the apo forms of ferrichrome in the micromolar range and cyclic voltammetry reveals the presence of redox-Bohr effect, which broadens the range of ferric-siderophore substrates that can be thermodynamically accessible for reduction. This study suggests that despite the structural differences between FSR and SIP proteins, mechanistic similarities exist between the two classes of proteins.

How Thermophilic Gram-Positive Organisms Perform Extracellular Electron Transfer: Characterization of the Cell Surface Terminal Reductase OcwA.

Extracellular electron transfer is the key process underpinning the development of bioelectrochemical systems for the production of energy or added-value compounds. Thermincola potens JR is a promising Gram-positive bacterium to be used in these systems because it is thermophilic. In this paper, we describe the structural and functional properties of the nonaheme cytochrome OcwA, which is the terminal reductase of this organism. The structure of OcwA, determined at 2.2-Å resolution, shows that the overall fold and organization of the hemes are not related to other metal reductases and instead are similar to those of multiheme cytochromes involved in the biogeochemical cycles of nitrogen and sulfur. We show that, in addition to solid electron acceptors, OcwA can also reduce soluble electron shuttles and oxyanions. These data reveal that OcwA can work as a multipurpose respiratory enzyme allowing this organism to grow in environments with rapidly changing availability of terminal electron acceptors without the need for transcriptional regulation and protein synthesis.IMPORTANCE Thermophilic Gram-positive organisms were recently shown to be a promising class of organisms to be used in bioelectrochemical systems for the production of electrical energy. These organisms present a thick peptidoglycan layer that was thought to preclude them to perform extracellular electron transfer (i.e., exchange catabolic electrons with solid electron acceptors outside the cell). In this paper, we describe the structure and functional mechanisms of the multiheme cytochrome OcwA, the terminal reductase of the Gram-positive bacterium Thermincola potens JR found at the cell surface of this organism. The results presented here show that this protein can take the role of a respiratory "Swiss Army knife," allowing this organism to grow in environments with soluble and insoluble substrates. Moreover, it is shown that it is unrelated to terminal reductases found at the cell surface of other electroactive organisms. Instead, OcwA is similar to terminal reductases of soluble electron acceptors. Our data reveal that terminal oxidoreductases of soluble and insoluble substrates are evolutionarily related, providing novel insights into the evolutionary pathway of multiheme cytochromes.

Nature of the Surface-Exposed Cytochrome-Electrode Interactions in Electroactive Biofilms of Desulfuromonas acetoxidans.

Metal-respiring bacteria are microorganisms capable of oxidizing organic pollutants present in wastewater and transferring the liberated electrons to an electrode. This ability has led to their application as catalysts in bioelectrochemical systems (BESs), a sustainable technology coupling bioremediation to electricity production. Crucial for the functioning of these BESs is a complex protein architecture consisting of several surface-exposed multiheme proteins, called outer membrane cytochromes, wiring the cell metabolism to the electrode. Although the role of these proteins has been increasingly understood, little is known about the protein-electrode interactions and their impact on the performance of BESs. In this study, we used surface-enhanced resonance Raman spectroscopy in combination with electrochemical techniques to unravel the nature of the protein-electrode interaction for the outer membrane cytochrome OmcB from Desulfuromonas acetoxidans (Dace). Comparing the spectroelectrochemical properties of OmcB bound directly to the electrode surface with those of the same protein embedded inside an electroactive biofilm, we have shown that the surface-exposed cytochromes of Dace biofilms are in direct contact with the electrode surface. Even if direct binding causes protein denaturation, the biofilm possesses the ability to minimize the extent of the damage maximizing the amount of cells in direct electrical communication with the electrode.

Heterologous expression and purification of a multiheme cytochrome from a Gram-positive bacterium capable of performing extracellular respiration.

Microbial electrochemical technologies are emerging as environmentally friendly biotechnological processes. Recently, a thermophilic Gram-positive bacterium capable of electricity production in a microbial fuel cell was isolated. Thermincola potens JR contains several multiheme c-type cytochromes that were implicated in the process of electricity production. In order to understand the molecular basis by which Gram-positive bacteria perform extracellular electron transfer, the relevant proteins need to be characterized in detail. Towards this end, a chimeric gene containing the signal peptide from Shewanella oneidensis MR-1 small tetraheme cytochrome c (STC) and the gene sequence of the target protein TherJR_0333 was constructed. This manuscript reports the successful expression of this chimeric gene in the Gram-negative bacterium Escherichia coli and its subsequent purification and characterization. This methodology opens the possibility to study other multiheme cytochromes from Gram-positive bacteria, allowing the extracellular electron transfer mechanisms of this class of organisms to be unraveled.

Analysis of the residual alignment of a paramagnetic multiheme cytochrome by NMR.

Residual dipolar couplings measured by NMR spectroscopy reveal that the rhombicity of the electronic structure of low-spin paramagnetic hemes determines their relative contribution to the preferential orientation of a protein with multiple hemes when placed in a strong magnetic field.

Biofunctionalized nanoparticles with pH-responsive and cell penetrating blocks for gene delivery.

Bridging the gap between nanoparticulate delivery systems and translational gene therapy is a long sought after requirement in nanomedicine-based applications. However, recent developments regarding nanoparticle functionalization have brought forward the ability to synthesize materials with biofunctional moieties that mimic the evolved features of viral particles. Herein we report the versatile conjugation of both cell penetrating arginine and pH-responsive histidine moieties into the chitosan polymeric backbone, to improve the physicochemical characteristics of the native material. Amino acid coupling was confirmed by 2D TOCSY NMR and Fourier transform infrared spectroscopy. The synthesized chitosan-histidine-arginine (CH-H-R) polymer complexed plasmid DNA biopharmaceuticals, and spontaneously assembled into stable 105 nm nanoparticles with spherical morphology and positive surface charge. The functionalized delivery systems were efficiently internalized into the intracellular compartment, and exhibited remarkably higher transfection efficiency than unmodified chitosan without causing any cytotoxic effect. Additional findings regarding intracellular trafficking events reveal their preferential escape from degradative lysosomal pathways and nuclear localization. Overall, this assembly of nanocarriers with bioinspired moieties provides the foundations for the design of efficient and customizable materials for cancer gene therapy.

Crystallization and preliminary crystallographic studies of FoxE from Rhodobacter ferrooxidans SW2, an Fe(II) oxidoreductase involved in photoferrotrophy.

FoxE is a protein encoded by the foxEYZ operon of Rhodobacter ferrooxidans SW2 that is involved in Fe(II)-based anoxygenic photosynthesis (`photoferrotrophy'). It is thought to reside in the periplasm, where it stimulates light-dependent Fe(II) oxidation. It contains 259 residues, including two haem c-binding motifs. As no three-dimensional model is available and there is no structure with a similar sequence, crystals of FoxE were produced. They diffracted to 2.44 Å resolution using synchrotron radiation at the Fe edge. The phase problem was solved by SAD using SHELXC/D/E and the experimental maps confirmed the presence of two haems per molecule.

Conformational component in the coupled transfer of multiple electrons and protons in a monomeric tetraheme cytochrome.

Cell metabolism relies on energy transduction usually performed by complex membrane-spanning proteins that couple different chemical processes, e.g. electron and proton transfer in proton-pumps. There is great interest in determining at the molecular level the structural details that control these energy transduction events, particularly those involving multiple electrons and protons, because tight control is required to avoid the production of dangerous reactive intermediates. Tetraheme cytochrome c(3) is a small soluble and monomeric protein that performs a central step in the bioenergetic metabolism of sulfate reducing bacteria, termed "proton-thrusting," linking the oxidation of molecular hydrogen with the reduction of sulfate. The mechano-chemical coupling involved in the transfer of multiple electrons and protons in cytochrome c(3) from Desulfovibrio desulfuricans ATCC 27774 is described using results derived from the microscopic thermodynamic characterization of the redox and acid-base centers involved, crystallographic studies in the oxidized and reduced states of the cytochrome, and theoretical studies of the redox and acid-base transitions. This proton-assisted two-electron step involves very small, localized structural changes that are sufficient to generate the complex network of functional cooperativities leading to energy transduction, while using molecular mechanisms distinct from those established for other Desulfovibrio sp. cytochromes from the same structural family.

Cooperativity between electrons and protons in a monomeric cytochrome c(3): the importance of mechano-chemical coupling for energy transduction.

To fully understand the structural bases for the mechanisms of biological energy transduction, it is essential to determine the microscopic thermodynamic parameters which describe the properties of each centre involved in the reactions, as well as its interactions with the others. These interactions between centres can then be interpreted in the light of structural features of the proteins. Redox titrations of cytochrome c(3) from Desulfovibrio desulfuricans ATCC 27774 followed by NMR and visible spectroscopy were analysed by using an equilibrium thermodynamic model. The network of homotropic and heterotropic cooperativities results in the coupled transfer of electrons and protons under physiological conditions. The microscopic characterisation allows the identification of several pairs of centres for which there are clear conformational (non-Coulombic) contributions to their coupling energies, thus establishing the existence of localised redox- and acid-base-linked structural modifications in the protein (mechano-chemical coupling). The modulation of interactions between centres observed for this cytochrome may be an important general phenomenon and is discussed in the framework of its physiological function and of the current focus of energy transduction research.

Comparative redox and pKa calculations on cytochrome c3 from several Desulfovibrio species using continuum electrostatic methods.

A comparative study of the pH-dependent redox mechanisms of several members of the cytochrome c3 family has been carried out. In a previous work, the molecular determinants of this dependency (the so-called redox-Bohr effect) were investigated for one species using continuum electrostatic methods to find groups with a titrating range and strength of interaction compatible with a mediating role in the redox-Bohr effect. Here we clarify these aspects in the light of new and improved pKa calculations, our findings supporting the hypothesis of propionate D from heme I being the main effector in the pH-dependent modulation of the cytochrome c3 redox potentials in all the c3 molecules studied here. However, the weaker (but significant) role of other titrating groups cannot be excluded, their importance and identity changing with the particular molecule under study. We also calculate the relative redox potentials of the four heme centers among the selected members of the c3 family, using a continuum electrostatic method that takes into account both solvation and interaction effects. Comparison of the calculated values with available data for the microscopic redox potentials was undertaken, the quality of the agreement being dependent upon the choice of the dielectric constant for the protein interior. We find that high dielectric constants give best correlations, while low values result in better magnitudes for the calculated potentials. The possibility that the crystallographic calcium ion in c3 from Desulfovibrio gigas may be present in the solution structure was tested, and found to be likely.

Functional and mechanistic studies of cytochrome c3 from Desulfovibrio gigas: thermodynamics of a "proton thruster".

Nuclear magnetic resonance and visible spectroscopies were used to determine the thermodynamic parameters of the four hemes in cytochrome c3 from Desulfovibrio gigas at 298 and 277 K and to investigate the mechanism of electron/proton energy transduction. Data obtained in the pH range from 5 to 9 were analyzed according to a model in which the hemes interact with each other (redox cooperativities) and with an ionizable center (redox-Bohr cooperativities). The results obtained at the two temperatures allow the deconvolution of the entropic contribution to the free energy of the four hemes, to the acid-base equilibrium of the ionizable center, and to the network of cooperativities among the five centers. The redox potentials of the hemes are modulated by the enthalpic contribution to the free energy, and evidence for the participation of the propionates of heme I in the redox-Bohr effect is presented. The network of interactions between the centers in this protein facilitates the concerted transfer of electrons and protons, in agreement with the "proton thruster" mechanism proposed for electronic to protonic energy transduction by cytochromes c3.

Determination of solution structures of paramagnetic proteins by NMR.

Standard procedures for using nuclear Overhauser enhancements (NOE) between protons to generate structures for diamagnetic proteins in solution from NMR data may be supplemented by using dipolar shifts if the protein is paramagnetic. This is advantageous since the electron -nuclear dipolar coupling provides relatively long-range geometric information with respect to the paramagnetic centre which complements the short-range distance constraints NOEs. Several different strategies have been developed to date, but none of these attempts to combine data from NOEs and dipolar shifts in the initial stages of structure calculation or to determine three dimensional protein structures together with their magnetic properties. This work shows that the magnetic and atomic structures are highly correlated and that it is important to have additional constraints both to provide starting parameters for the magnetic properties and to improve the definition of the best fit. Useful parameters can be obtained for haem proteins from Fermi contact shifts; this approach is compared with a new method based on the analysis of dipolar shifts in haem methyl groups with respect to data from horse and tuna ferricytochromes c. The methods developed for using data from NOEs and dipolar shifts have been incorporated in a new computer program, PARADYANA, which is demonstrated in application to a model data set for the sequence of the haem octapeptide known as microperoxidase-8.

Structural and functional characterization of cytochrome c3 from D. desulfuricans ATCC 27774 by 1H-NMR.

Cooperativity between redox and protonation centres is known to be crucial for the function of complex proteins, but it is often difficult to describe in terms of thermodynamic parameters. Cytochrome c3 is a good model for these studies since, while retaining the overall complexity of larger systems, it is suitable for detailed crystallographic and spectroscopic studies. Assignment of the haem substituent NMR resonances, together with NMR redox titrations of cytochrome c3 from D. desulfuricans ATCC 27774, was used to correlate relative redox potentials to specific haems in the structure: haem II approximately equal to haem I < haem IV < haem III. This order is different from that determined for the homologous proteins studied and in disagreement with that previously reported for this cytochrome (Morais, J., Palma, N., Frazäo, C., Caldeira, J., LeGall, J., Moura, I., Moura, J.J.G. and Carrondo, M.A. (1995) Biochemistry 34, 12830-12841).