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 ...

No abstract

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.

SummaryA 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.We obtained crystal and solution structures of NDH-2 from S. aureus, showing that it is a dimer in solution. We report fast kinetic analyses of the protein and detected a charge-transfer complex formed between NAD + and the reduced flavin, which is dissociated by the quinone. We observed that the quinone reduction is the rate limiting step and also the only half-reaction affected by the presence of HQNO, an inhibitor. We analyzed protein-substrate interactions by fluorescence and STD-NMR spectroscopies, which indicate that NADH and the quinone bind to different sites. In summary, our combined results show the presence of distinct binding sites for the two substrates, identified quinone reduction as the rate limiting step and indicate the establishment of a NAD + -protein complex, which is released by the quinone.

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.

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 mechanochemical 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.Recent developments in techniques of structural biology have opened the way for probing the mechanisms used by biological macromolecules involved in energy transduction at the molecular level. The structural analysis of bacteriorhodopsin trapped in the M photointermediate state (1), the structures in the oxidized and reduced forms of cytochromes c 3 that perform a coupled two-electron step associated with proton transfer (2, 3), and the establishment of the coupled transfer of electrons and protons to the 3Fe-4S cluster of Azotobacter vinelandii ferredoxin (4) are just a few recent examples where results from different techniques are integrated in a description at the atomic level of the energy-transducing events. The phenomenon of energy transduction relies on coupled events (5), whether they involve only electrostatic interactions or structural rearrangements of the active sites or its surroundings (mechano-chemical coupling), which may be more important than the electrostatic component of the overall coupling (6). The pumping of proton(s) at the beginning of re-reduction of cytochrome c oxidase (7) can be described using a model in which the electrostatic attraction of electrons and protons is overcome (8), a situation that requires structural changes involving charged residues. Small proteins capable of performing energy transduction provide easier access to the structural bases for the underlying mechanisms, as the recent advances in the understanding of the proton pumping by the 26-kDa bacteriorhodopsin demonstrate (1, 9).This wo...

A thermodynamic model is presented to describe the redox behaviour of the tetraheme cytochrome c3 from Desu(fbvihrio gigns. This molecule displays different intrinsic redox potentials for the four hemes and during thc redox titration process, interactions among different hemes occur, thus altering the values of redox potentials according to which of the hemes are oxidized [Santos, H., Moura, J. J. G., Moura, I., LeGall Chem. 245, 6241 -62501 coupled to a proton-linked equilibrium between two tertiary structures. Such a formalism, which requires a reduced number of parameters, is able to fully account quantitatively for the pH dependence of the NMK redox-titration curves. The 'redox-Bohr' effcct is discussed in terms of the available structure and thermodynamic data and a functional mechanism is proposed.Cytochrome c3 from De.su&vihrio gigas is a monomeric tetraheme protein which displays an oxidation/reduction equilibrium characterized by having hemes with different (and pH dependent) midpoint redox potentials. Moreover, during the redox process they are affected by the oxidation state of the surrounding hemes (interacting potentials), as demonstrated by previous N M R studies [I]. The cooperativity network resulting from these interacting potentials, has been previously discussed in terms of the functional properties of this electrontransfer protein [2]. In addition, recent observations by resonance-Raman [3] and EPK spectroscopy [4, 51 have indeed supported a similar behaviour for the redox-titration curve in cytochrome c3 from different sources.Although the X-ray structures of cytochrome c3 from D . gig0.r and from closely related and similarly behaving cytochromes have bcen resolved to high resolution [6 -81, providing evidence of the close spatial relationship of the four hemes, the detailed physical and structural mechanism underlying such a cooperative beliaviour has not been clarified up to now.It has been pointed out that electron transfer can take place over large distances through protein interiors [9-1 I]. This concept implies that changes in the tertiary conformation of the protein moiety can be very important in modulating the mutual influence of two redox centers within the same protein. In this way, the effectiveness of a preferential pathway for the electron transfer between two redox centers through the protein can be either magnified or dissipated by conformational changes of the tertiary structural arrangement. Such a mechanism can become very important whenever a relatively However, the presence of four functionally interacting hemes makes the phenomenological analysis of the redoxequilibrium process very complicated even for a single pH value, requiring the introduction of 16 pH-dependent binding parameters whose physical and numerical significance cannot be easily characterized [l]. 'This prompted us to elaborate a model according to a formalism, which follows from the interaction network IzH4 [12] coupled to an equilibrium bctween two tertiary structures, to carry out a more straightfor...

No abstract