NMR studies of electron transfer mechanisms in a protein with interacting redox centres: Desulfovibrio gigas cytochrome c₃

Abstract

The proton NMR spectra of the tetrahaem cytochrome c3 from Desulfovibrio gigas were examined while varying the pH and the redox potential. The analysis of the N M R reoxidation pattern was based on a model for the electron distribution between the four haems that takes into account haem-haem redox interactions. The intramolecular electron exchange is fast on the N M R time scale (larger than lo5 s-I ) . The NMR data concerning the pH dependence of the chemical shift of haem methyl resonances in different oxidation steps and resonance intensities are not compatible with a non-interacting model and can be explained assuming a redox interaction between the haems. A complete analysis at pH* = 7.2 and 9.6, shows that the haem-haem interacting potentials cover a range from -50 mV to +60 mV. The midpoint redox potentials of some of the haems, as well as some of their interacting potentials, are pH-dependent. The physiological relevance of the modulation of the haem midpoint redox potentials by both the pH and the redox potential of the solution is discussed.Cy tochromc c3 ( M , 13 000) is a multihaem protein found in anaerobic sulfate-reducing bacteria belonging to the genus Desulfuvibrin. Each molecule contains four haems with an unusually low redox potential. They are covalently attached to the polypeptide chain by thioether linkages provided by cysteinyl residues and two histidines are used as axial ligands.Cytochrome [39] have been applied to elucidate the mechanism of electron transfer in cytochrome c3.The midpoint redox potentials of the four haems are different, in general. EPR measurements coupled with potentiometric titrations were performed to determine the midpoint redox potentials of the individual haems of D. gigas cytochrome c3 [27] (-235mV, -235mV, -306mV, -315mV) [35]; the experiments were fitted by digital simulations and the best fit was obtained with -220mV, -272mV, -292mV, and --310mV, for the four macroscopic midpoint redox potentials. Bianco et al. [36] reported values of -170mV, -310mV, -360mV and -400mV for the half-wave potentials of cytochrome c3 from D. desidfuricans (Norway 4). However. it should be emphasized that the individual potentials obtained from electrochemical studies are macroscopic rather than microscopic parameters and can not be compared directly with the values obtained from EPR measurements. Furthermore, thc EPR measurements

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

“…The haems are covalently bound to the polypeptide chain by two thioether bridges and bis-histidinyl axial coordination and have very low redox potentials. For the D. gigas and D. vulgaris cytochromes, the microscopic haem mid-point redox potentials were shown to be dependent on the redox state of the protein and pH [3][4][5][6], thus establishing a network of redox and redox-Bohr cooperativities. In the case of D. vulgaris, this was used to explain how cytochrome c3 can act as a charge separation device for electrons and protons originating from the oxidation of molecular hydrogen via hydrogenase, leading to energy transduction [7].…”

“…When the intermolecular electron exchange is slow on the NMR time scale and the intramolecular electron exchange is fast [3,4], a separate set of NMR resonances is observed for the haem protons in each stage of oxidation (numbered according to the number of oxidised haems) with shifts which depend on the relative populations of each oxidised haem. This property is extremely useful since it can not only be used to determine the relative microscopic redox potentials for the different haems [3], but also provides the means to extend the structure-specific assignments from the spectra of the reduced form by the observation of chemical exchange connectivities between resonances of the same haem nucleus in the different stages of oxidation of the protein [3,4,[20][21][22]. In multiredox-centre proteins, this is a crucial piece of information for understanding their mode of action since it links unequivocally specific structural features with the respective redox potentials.…”

Solution structure of Desulfovibrio vulgaris (Hildenborough) ferrocytochrome c 3 : structural basis for functional cooperativity 1 1Edited by P. E. Wright

“…This is still the case for the tetrahaem cytochromes c 3 from Desulfovibrio spp., even though considerable progress has been made in rationalising their thermodynamic properties (Santos et al, 1984;Coletta et al, 1991;Turner et al, 1994Turner et al, , 1996Park et al, 1996;Louro et al, 1996;Salgueiro et al, 1997) and high-resolution X-ray structures are available for several cytochromes c 3 in the oxidised form (Higuchi et al, 1984;Czjzek et al, 1994;Matias et al, 1996;Simo Ä es et al, 1998). Tetrahaem cytochrome c 3 from Desulfovibrio vulgaris (Hildenborough: DvHc 3 ) is a small (14 kDa; 107 amino acid residues) soluble protein whose crystal structure in the oxidised state has been determined by X-ray diffraction (Matias et al, 1993;Simo Ä es et al, 1998).…”

Electrochemical study of an electron shuttle diheme protein: The cytochrome c from T. thermophilus

“…Nonetheless, the EPR titrations suggest that the potentiometric properties of the individual hemes are divergent in the fragments and full-length protein and they demonstrate the existence of cooperative interactions between them. This behavior was also suggested in other multi redox center proteins, including diheme cyt c4 [33], cyt c oxidase [47,48] and tetraheme cyt c3 [49,50]. Interestingly, as a consequence of the interaction scheme, the N-terminal domain is easier to reduce than the C-terminal domain in the full-length protein.…”