Teresa Catarino

Cooperativity between electrons and protons in a monomeric Cytochrome c3: 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 c3 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.

Redox-Bohr effect in electron/proton energy transduction: cytochrome c3 coupled to hydrogenase works as a 'proton thruster' in Desulfovibrio vulgaris

Abstract A central step in the metabolism of Desulfovibrio spp. is the oxidation of molecular hydrogen catalyzed by a periplasmic hydrogenase. However, this enzymatic activity is quite low at physiological pH. The hypothesis that, in the presence of the tetrahaem cytochrome c3, hydrogenase can maintain full activity at physiological pH through the concerted capture of the resulting electrons and protons by the cytochrome was tested for the case of Desulfovibrio vulgaris (Hildenborough). The crucial step involves an electron-to-proton energy transduction, and is achieved through a network of cooperativities between redox and ionizable centers within the cytochrome (redox-Bohr effect). This mechanism, which requires a relocation of the proposed proton channel in the hydrogenase structure, is similar to that proposed for the transmembrane proton pumps, and is the first example which shows evidence of functional energy transduction in the absence of a membrane confinement.

Redox-Bohr effect in the tetrahaem cytochrome c3 from Desulfovibrio vulgaris: a model for energy transduction mechanisms

Abstract Using potentiometric titrations, two protons were found to participate in the redox-Bohr effect observed for cytochrome c3 from Desulfovibrio vulgaris (Hildenborough). Within the framework of the thermodynamic model previously presented, this finding supports the occurrence of a concerted proton-assisted 2e– step, ideally suited for the coupling role of cytochrome c3 to hydrogenase. Furthermore, at physiological pH, it is shown that when sulfate-reducing bacteria use H2 as energy source, cytochrome c3 can be used as a charge separation device, achieving energy transduction by energising protons which can be left in the acidic periplasmic side and transferring deenergised electrons to sulfate respiration. This mechanism for energy transduction, using a full thermodynamic data set, is compared to that put forward to explain the proton-pumping function of cytochrome c oxidase.

Homotropic and heterotropic cooperativity in the tetrahaem cytochrome c3 from Desulfovibrio vulgaris.

The thermodynamic parameters which govern the homotropic (e-/e-) and heterotropic (e-/H+) cooperativity in the tetrahaem cytochrome c3 isolated from Desulfovibrio vulgaris (Hildenborough) were determined, using the paramagnetic shifts of haem methyl groups in the NMR spectra of intermediate oxidized states at different pH levels. A model is put forward to explain how the network of positive and negative cooperativities between the four haems and acid/base group(s) enables the protein to achieve a proton-assisted 2e- step.

Distance dependence of interactions between charged centres in proteins with common structural features

Data collected for interactions among redox centres, and interactions between redox centres and acid–base residues in a family of small multihaem cytochromes are analysed. The distance dependent attenuation of the interactions between non‐surface charges, for separations that range from 8 to 23 Å, can be described by a simple function derived from the Debye–Hückel formalism, fit to 9.5 and 7.6 as values for the relative dielectric constant and Debye length, respectively. However, there is considerable scatter in the data despite the structural similarities among the proteins, which is discussed in the framework of using such simple models in predicting properties of novel proteins.

Type‐II NADH:quinone oxidoreductase from Staphylococcus aureus has two distinct binding sites and is rate limited by quinone reduction

A prerequisite for any rational drug design strategy is understanding the mode of protein–ligand interaction. This motivated us to explore protein–substrate interaction in Type‐II NADH:quinone oxidoreductase (NDH‐2) from Staphylococcus aureus, a worldwide problem in clinical medicine due to its multiple drug resistant forms. NDHs‐2 are involved in respiratory chains and recognized as suitable targets for novel antimicrobial therapies, as these are the only enzymes with NADH:quinone oxidoreductase activity expressed in many pathogenic organisms.

Thermodynamic Control of Electron Transfer Rates in Multicentre Redox Proteins

In the analysis of kinetic data from multicentre redox proteins, it is essential to distinguish between the observable macroscopic rate constants and the structurally relevant microscopic properties. This distinction is complicated by the existence of interactions between centres. The problem is illustrated by the case of two interacting redox centres and generalised for the analysis of stopped‐flow kinetic data for the reduction of cytochrome c3, in which four redox centres and at least one proteolytic centre are mutually interacting. It is shown that fast intramolecular electron transfer, which is typical of many multicentre redox proteins, and, where present, fast proton exchange, ensure that only N rate constants can be measured for a protein with N redox centres. The equations that relate the observable macroscopic rate constants to the microscopic rate constants of individual centres depend on a set of parameters that can be approximated by using the Marcus theory of electron transfer together with a set of reasonable assumptions. The results are tested by fitting experimental data for the reduction of cytochrome c3 by sodium dithionite, including its pH dependence.

Evidence for interaction between the triheme cytochrome PpcA from Geobacter sulfurreducens and anthrahydroquinone-2,6-disulfonate, an analog of the redox active components of humic substances.

The bacterium Geobacter sulfurreducens displays an extraordinary respiratory versatility underpinning the diversity of electron donors and acceptors that can be used to sustain anaerobic growth. Remarkably, G. sulfurreducens can also use as electron donors the reduced forms of some acceptors, such as the humic substance analog anthraquinone-2,6-disulfonate (AQDS), a feature that confers environmentally competitive advantages to the organism. Using UV-visible and stopped-flow kinetic measurements we demonstrate that there is electron exchange between the triheme cytochrome PpcA from Gs and AQDS. 2D-(1)H-(15)N HSQC NMR spectra were recorded for (15)N-enriched PpcA samples, in the absence and presence of AQDS. Chemical shift perturbation measurements, at increasing concentration of AQDS, were used to probe the interaction region and to measure the binding affinity of the PpcA-AQDS complex. The perturbations on the NMR signals corresponding to the PpcA backbone NH and heme substituents showed that the region around heme IV interacts with AQDS through the formation of a complex with a definite life time in the NMR time scale. The comparison of the NMR data obtained for PpcA in the presence and absence of AQDS showed that the interaction is reversible. Overall, this study provides for the first time a clear illustration of the formation of an electron transfer complex between AQDS and a G. sulfurreducens triheme cytochrome, shedding light on the electron transfer pathways underlying the microbial oxidation of humics.

Probing the Chemotaxis Periplasmic Sensor Domains from Geobacter sulfurreducens by Combined Resonance Raman and Molecular Dynamic Approaches: NO and CO Sensing

The periplasmic sensor domains encoded by genes gsu0582 and gsu0935 are part of methyl accepting chemotaxis proteins in the bacterium Geobacter sulfurreducens (Gs). The sensor domains of these proteins contain a heme-c prosthetic group and a PAS-like fold as revealed by their crystal structures. Biophysical studies of the two domains showed that nitric oxide (NO) binds to the heme in both the ferric and ferrous forms, whereas carbon monoxide (CO) binds only to the reduced form. In order to address these exogenous molecules as possible physiological ligands, binding studies and resonance Raman (RR) spectroscopic characterization of the respective CO and NO adducts were performed in this work. In the absence of exogenous ligands, typical RR frequencies of five-coordinated (5c) high-spin and six-coordinated (6c) low-spin species were observed in the oxidized form. In the reduced state, only frequencies corresponding to the latter were detected. In both sensors, CO binding yields 6c low-spin adducts by replacing the endogenous distal ligand. The binding of NO by the two proteins causes partial disruption of the proximal Fe-His bond, as revealed by the RR fingerprint features of 5cFe-NO and 6cNO-Fe-His species. The measured CO and NO dissociation constants of ferrous GSU0582 and GSU0935 sensors reveal that both proteins have high and similar affinity toward these molecules (K(d) approximately = 0.04-0.08 microM). On the contrary, in the ferric form, sensor GSU0582 showed a much higher affinity for NO (K(d) approximately = 0.3 microM for GSU0582 versus 17 microM for GSU0935). Molecular dynamics calculations revealed a more open heme pocket in GSU0935, which could account for the different affinities for NO. Taken together, spectroscopic data and MD calculations revealed subtle differences in the binding properties and structural features of formed CO and NO adducts, but also indicated a possibility that a (5c) high-spin/(6c) low-spin redox-linked equilibrium could drive the physiological sensing of Gs cells.

Relative importance of driving force and electrostatic interactions in the reduction of multihaem cytochromes by small molecules

Multihaem cytochromes are essential to the energetics of organisms capable of bioremediation and energy production. The haems in several of these cytochromes have been discriminated thermodynamically and their individual rates of reduction by small electron donors were characterized. The kinetic characterization of individual haems used the Marcus theory of electron transfer and assumed that the rates of reduction of each haem by sodium dithionite depend only on the driving force, while electrostatic interactions were neglected. To determine the relative importance of these factors in controlling the rates, we studied the effect of ionic strength on the redox potential and the rate of reduction by dithionite of native Methylophilus methylotrophus cytochrome c″ and three mutants at different pH values. We found that the main factor determining the rate is the driving force and that Marcus theory describes this satisfactorily. This validates the method of the simultaneous fitting of kinetic and thermodynamic data in multihaem cytochromes and opens the way for further investigation into the mechanisms of these proteins.