NMR Studies of Cooperativity in the Tetrahaem Cytochrome c₃ from Desulfovibrio vulgaris

Abstract

The thermodynamic properties of the Desulfovibrio vulgaris (Hildenborough) tetrahaem cytochrome c3 (Dvc,) are rationalised by a model which involves both homotropic (e-/e-) and heterotropic (e-/H+) cooperativity. The paramagnetic shifts of a methyl group from each haem of the DVC, have been determined in each stage of oxidation at several pH values by means of two-dimensional exchange NMR. The thermodynamic parameters are obtained by fitting the model to the NMR data and to redox titrations followed by visible spectroscopy. They show significant positive cooperativity between two of the haems whereas the remaining interactions appear to be largely electrostatic in origin. These parameters imply that the protein undergoes a proton-assisted two-electron transfer which can be used for energy transduction. Comparison with the crystal structure together with measurement of the kinetics of proton exchange suggest that the pH dependence is mediated by a charged residue(s) readily acessible to the solvent and close to haem I.Keywords: cooperativity ; energy transduction ; multiheme cytochrome ; NMR ; redox-Bohr.The functional cooperativity between different regions of some proteins [ l ] is a fundamental property to control and coordinate important chemical events in the living cell. Although the molecular basis for the fine regulation of several types of cooperativity mechanisms has been successfully established [2], little is known about the structural basis for electron/electron and electron/proton cooperativities and their role either in electron transfer or in energy transduction [3].Desulfovibrio spp. cytochrome c, is a small (=14 m a ) , monomeric tetrahaem protein which exhibits cooperativity between the four haems and acidmase group(s): the haem redox potentials are pH dependent (redox-Bohr effect) and each haem redox potential is dependent on the oxidation state of the other three haems (redox interaction potentials) [4-61. Due to its small size and the fact that the haems are diamagnetic in the reduced state and paramagnetic in the oxidised one, NMR is particularly well suited to characterise this protein from the structural and thermodynamic point of view [4,[7][8][9][10][11][12][13][14][15].Furthermore, several X-ray structures are available for cytochromes c3 from Desulfovibrio spp. [16-231.The thermodynamic properties of cytochrome c, have been analysed by previous NMR studies [4,5,8,15, 241. In the first of these studies an NMR data set obtained at two discrete pH values for Desulfovibrio gigas cytochrome c, was used to calculate nine parameters (three relative microscopic redox potentials and six haem-haem redox interactions) for each pH value, independently treated. The redox interaction potentials were fixed according to the maximum concentration reached by the intermediate oxidation stages (defined according to the number of oxidised haems) in redox titrations followed by NMR [4]. Using the same NMR data set, a second study proposed a model with 21 parameters in which the four haem redox potentials ...

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

“…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). Previous studies have shown that the four haem groups form a complex network of interacting redox centres, resulting in homotropic or redox cooperativity between their redox potentials (Turner et al, 1996). The potentials are also dependent on solution pH, implying heterotropic or redox-Bohr cooperativity between their haem redox and protonation energies Turner et al, 1996).…”

Effect of Hydrogen-Bond Networks in Controlling Reduction Potentials in Desulfovibrio vulgaris (Hildenborough) Cytochrome c3 Probed by Site-Specific Mutagenesis

“…In T24VDVHc 3 , differences caused near heme III by the mutation could be responsible for this small variation, through the modulation of the dielectric constant or movement of the ionizable group. Previous work showed that the redox-Bohr effect in DVHc 3 is controlled by heme I propionates and neighboring residues (11,27,28), and since the residue mutated is not located close to heme I, the four macroscopic reduction potentials have a similar pH dependence in both cytochromes, although it is shifted to significantly higher values in T24VDVHc 3 ( Figure 5).…”

“…Since DVHc 3 exhibits fast intramolecular and slow intermolecular electron exchange on the NMR time scale, any heme substituent displays five discrete NMR signals corresponding to each of the five possible macroscopic oxidation stages, connected by four one-electron steps. Thus, the paramagnetic shifts of heme substituents can be used to obtain the relative microscopic reduction potentials of the heme groups in each stage of oxidation (10,11,36). However, the paramagnetic shift observed for a particular heme substituent depends not only on the Fermi contact and the dipolar contributions of its own heme (intrinsic shift) but also on the dipolar shift due to the other hemes (extrinsic shift) (36)(37)(38).…”

“…The process of fitting the thermodynamic parameters involves adjusting the energies, and hence the populations, of the 32 microstates in which each of the four hemes is oxidized or reduced and the ionizable center is protonated or deprotonated (11). Since proton exchange is fast on the NMR time scale, the properties of the hemes are represented by the population of pairs of microstates, which we label in the next section with the numbers of those hemes that are oxidized in each case.…”

“…Together, the positive redox-Bohr effect and the positive redox cooperativity between hemes I and II explain the effect of cytochrome c 3 in stimulating hydrogenase activity (H 2 f 2H + + 2e -) (11).…”