Browsing by Author "Turner, David"
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- Electronic Structure of Low-Spin Ferric Porphyrins: 13C NMR Studies of the Influence of Axial Ligand OrientationPublication . Louro, Ricardo; Correia, I. J.; Brennan, Lorraine; Coutinho, Isabel; Xavier, António; Turner, DavidHeteronuclear multiple quantum NMR is used to measure the paramagnetic 13C shifts of the α substituents of the hemes in five different tetraheme ferricytochromes c3. The shifts of the 20 bis-histidine ligated hemes are assigned and then analyzed in terms of a model based on the π molecular orbitals of the heme under perturbed D4 symmetry, which yields the orientation of the rhombic perturbation, θ, and an energy splitting parameter, ΔE. Comparison of these parameters with crystal structures provides a test of the nature and extent of the influence of axial ligand orientation on the electronic structure of the heme. Despite possible differences between structures in solution and in the crystal, a clear correlation is found between θ and the resultant of the normals to the imidazole planes, and between ΔE and the angle between the normals. A weaker dependence of ΔE upon θ is also apparent. This is analogous to the results of low-temperature EPR studies of model compounds, which have been attributed to pseudo-Jahn−Teller distortion of the porphyrin. However, the effect is also predicted by extended Hückel calculations made with undistorted geometries. This work demonstrates that the variation in the electronic structure of bis-histidinyl hemes c is dominated by the geometry of the axial ligands and that other perturbations, such as asymmetric substitution of the porphyrin or low symmetry of the surrounding protein, are relatively minor. The correlations with θ and ΔE can, therefore, be used to determine the ligand geometry with sufficient accuracy to detect differences between structures in solution and in the crystal. The analysis can also be used to locate the principal axes of the magnetic susceptibility tensors of ferrihemes as well as providing orientational constraints for the axial ligands for the calculation of solution structures of paramagnetic proteins. This is particularly important since paramagnetic relaxation may make it impossible to observe NOE effects to the imidazole protons, leaving the geometry of the heme pocket poorly defined.
- Proton-assisted Two-electron Transfer in Natural Variants of Tetraheme Cytochromes from Desulfomicrobium Sp.Publication . Correia, Ilídio Joaquim Sobreira; Paquete, Catarina; Coelho, Ana; Almeida, Claudia; Catarino, Teresa; Louro, Ricardo; Frazão, Carlos; Saraiva, Lígia M.; Carrondo, Maria; Turner, David; Xavier, AntónioThe tetraheme cytochrome c3 isolated from Desulfomicrobium baculatum (DSM 1743)(Dsmb) was cloned, and the sequence analysis showed that this cytochrome differs in just three amino acid residues from the cytochrome c3 isolated from Desulfomicrobium norvegicum (Dsmn): (DsmnXXDsmb) Thr-37 → Ser, Val-45 → Ala, and Phe-88 → Tyr. X-ray crystallography was used to determine the structure of cytochrome c3 from Dsmb, showing that it is very similar to the published structure of cytochrome c3 from Dsmn. A detailed thermodynamic and kinetic characterization of these two tetraheme cytochromes c3 was performed by using NMR and visible spectroscopy. The results obtained show that the network of cooperativities between the redox and protonic centers is consistent with a synergetic process to stimulate the hydrogen uptake activity of hydrogenase. This is achieved by increasing the affinity of the cytochrome for protons through binding electrons and, reciprocally, by favoring a concerted two-electron transfer assisted by the binding of proton(s). The data were analyzed within the framework of the differences in the primary and tertiary structures of the two proteins, showing that residue 88, close to heme I, is the main cause for the differences in the microscopic thermodynamic parameters obtained for these two cytochromes c3. This comparison reveals how replacement of a single amino acid can tune the functional properties of energy-transducing proteins, so that they can be optimized to suit the bioenergetic constraints of specific habitats.
- Thermodynamic and kinetic characterization of trihaem cytochrome c3 from Desulfuromonas acetoxidansPublication . Correia, Ilídio Joaquim Sobreira; Paquete, Catarina; Louro, Ricardo; Catarino, Teresa; Turner, David; Xavier, AntónioTrihaem cytochrome c3 (also known as cytochrome c551.5 and cytochrome c7) is isolated from the periplasmic space of Desulfuromonas acetoxidans, a sulfur-reducing bacterium. Thermodynamic and kinetic data for the trihaem cytochrome c3 are presented and discussed in the context of the possible physiological implications of its functional properties with respect to the natural habitat of D. acetoxidans, namely as a symbiont with green sulfur bacteria working as a mini-sulfuretum. The thermodynamic properties were determined through the fit of redox titration data, followed by NMR and visible spectroscopy, to a model of four functional centres that describes the network of cooperativities between the three haems and one protolytic centre. The kinetics of trihaem cytochrome c3 reduction by sodium dithionite were studied using the stopped-flow technique and the data were fitted to a kinetic model that makes use of the thermodynamic properties to obtain the rate constants of the individual haems. This analysis indicates that the electrons enter the cytochrome mainly via haem I. The reduction potentials of the haems in this cytochrome show little variation with pH within the physiological range, and the kinetic studies show that the rates of reduction are also independent of pH in the range studied. Thus, although the trihaem cytochrome c3 is readily reduced by hydrogenases from Desulfovibrio sp. and its haem core is similar to that of the homologous tetrahaem cytochromes c3, its physico-chemical properties are quite different, which suggests that these multihaem cytochromes with similar structures perform different functions.
- Thermodynamic characterization of a tetrahaem cytochrome isolated from a facultative aerobic bacterium, Shewanella frigidimarina: a putative redox model for flavocytochrome c3Publication . Pessanha, Miguel; Louro, Ricardo; Correia, Ilídio Joaquim Sobreira; Rothery, Emma; Pankhurst, Kate; Reid, Graeme; Chapman, Stephen; Turner, David; Salgueiro, CarlosThe facultative aerobic bacterium Shewanella frigidimarina produces a small c-type tetrahaem cytochrome (86 residues) under anaerobic growth conditions. This protein is involved in the respiration of iron and shares 42% sequence identity with the N-terminal domain of a soluble flavocytochrome, isolated from the periplasm of the same bacterium, which also contains four c-type haem groups. The thermodynamic properties of the redox centres and of an ionizable centre in the tetrahaem cytochrome were determined using NMR and visible spectroscopy techniques. This is the first detailed thermodynamic study performed on a tetrahaem cytochrome isolated from a facultative aerobic bacterium and reveals that this protein presents unique features. The redox centres have negative and different redox potentials, which are modulated by redox interactions between the four haems (covering a range of 8—56mV) and by redox—Bohr interactions between the haems and an ionizable centre (-4 to −36mV) located in close proximity to haem III. All of the interactions between the five centres are clearly dominated by electrostatic effects and the microscopic reduction potential of haem III is the one most affected by the oxidation of the other haems and by the protonation state of the molecule. Altogether, this study indicates that the tetrahaem cytochrome isolated from S. frigidimarina (Sfc) has the thermodynamic properties to work as an electron wire between its redox partners. Considering the high degree of sequence identity between Sfc and the cytochrome domain of flavocytochrome c3, the structural similarities of the haem core, and that the macroscopic potentials are also identical, the results obtained in this work are rationalized in order to put forward a putative redox model for flavocytochrome c3.