In cytochromes c3 which contain four hemes per molecule, the redox properties of each heme may depend upon the redox state of the others. This effect can be described in terms of interaction redox potentials between the hemes and must be taken into account in the characterization of the redox properties of the molecule. We present here a method of measurement of these interactions based on the EPR study of the redox equilibria of the protein. The microscopic and macroscopic midpoint potentials and the interaction potentials are deduced from the analysis of the redox titration curves of the intensity and the amplitude of the EPR spectrum. This analysis includes a precise simulation of the spectrum of the protein in the oxidized state in order to determine the relative contribution of each heme to the spectral amplitude. Using our method on cytochrome c3 from D. desulfuricans Norway, we found evidence for the existence of weak interaction potentials between the hemes. The three interaction potentials which have been measured are characterized by absolute values lower than 20 mV in contrast with the values larger than 40-50 mV which have been reported for cytochrome c3 from D. gigas. Simulations of the spectra of samples poised at different potentials indicate a structural modification of the heme with the most negative potential during the first step of reduction. The correspondence between the redox sites as characterized by the EPR potentiometric titration and the hemes in the tridimensional structure is discussed.

We present a new examination of the EPR redox titration data for the tetraheme cytochrome c3 from Desulfovihrio vulgaris Miyazaki. Our analysis includes the contribution of the interaction potentials between the four redox sites and is based on the model previously developed for the study of cytochrome c3 from Desulfovihrio desulfuricans Norway. We observed, as for D. desulfuricans Norway cytochrome c3, that the conformation of the heme with the lowest redox potential, heme 4, is sensitive to the redox state of the heme with the highest potential, heme 1. However in D. vulgaris Miyazaki cytochrome c3 spectral simulations show that heme 4 is present in two conformational states which interconvert partially when heme 1 is reduced. The sets of redox parameters which satisfy the fitting procedure of the titration curves are in the following domain: -250 mV I ef I -220mV, -325mV I e2 I -320mV, -335mV 5 e3 5 -330mV, -360mV I e4 I -355mV, -5mV I I I 2 5 20mV, -10mV I I I 3 I 5mV, -15mV I I 2 3 5 -5mV, -15mV I I 2 4 I -10mV, -25mV I 134 I -15mV. As in D. desulfuricans Norway cytochrome c3 the interactions are moderate. Simple electrostatic considerations suggest that these moderate values could be related to the large accessibility of the hemes to the solvent. Our work does not confirm the existence of a cooperative interaction between heme 2 and heme 3 which has been proposed on the basis of electrochemical measurements.In biological electron carriers containing several redox sites, cooperative and anticooperative redox interactions could play a significant role in the activity. The tetraheme cytochrome c3, where the four redox sites are closely packed, appears as a good model for a study of these interactions. The three-dimensional structure is known for two different species, the cytochrome c3 from Desulfovihrio desulfuricans Norway (DdNc3) [l] and the cytochrome c3 from Desulfovihrio vulgaris Miyazaki (DvMc3) [2]. Precise calculations of electrostatic energies should then be possible in order to determine the main factors involved in the redox properties such as the accessibility to the solvent and the influence of the dipoles, permanent or induced, the ionized groups and the conformational variations accompanying the change of the redox state [3,4]. The present work is a part of our program concerning the experimental investigation of the redox properties of these systems [5 -71.The first data on redox interactions were obtained on cytochrome c3 from Desulfovihrio gigas (Dgc,) with an original method based on redox titrations followed by NMR spectroscopy [XI. They indicated the existence of cooperative redox interactions larger than + 40 mV and a pH dependence of the redox parameters. We have developed recently a different approach based on EPR redox titrations [7]. Application of our method to DdNc3 has shown that the interactions between hemes 2, 3 and 4 (numbered in order of decreasing redox potentials) are moderate, with absolute values smaller than 15 mV, and a spectrophotometric redox titration indicated no ...

We report the results of an EPR potentiometric titration of cytochrome c3 from DesulJbvibrio vulgaris Miyazaki: the EPR spectral features of the four hemes are identified. The four midpoint redox potentials, which are deduced from the integrated intensity variations as a function of the redox potential, are within the range -230 to -360 mV with two nearly equal intermediate values, in agreement with previous electrochemical measurements. A structural change of the environment of the heme with the most negative potential is observed during the first step of the reduction. The correspondence between the redox sites as characterized by the EPR potentiometric titration, and the hemes in the tridimensional structure, is discussed.

In cytochromes c3 which contain four hemes per molecule, the redox properties of each heme may depend upon the redox state of the others. This effect can be described in terms of interaction redox potentials between the hemes and must be taken into account in the characterization of the redox properties of the molecule. We present here a method of measurement of these interactions based on the EPR study of the redox equilibria of the protein. The microscopic and macroscopic midpoint potentials and the interaction potentials are deduced from the analysis of the redox titration curves of the intensity and the amplitude of the EPR spectrum. This analysis includes a precise simulation of the spectrum of the protein in the oxidized state in order to determine the relative contribution of each heme to the spectral amplitude. Using our method on cytochrome c3 from D. desuljuricans Norway, we found evidence for the existence of weak interaction potentials between the hemes. The three interaction potentials which have been measured are characterized by absolute values lower than 20 mV in contrast with the values larger than 40 -50 mV which have been reported for cytochrome c3 from D. gigas. Simulations of the spectra of samples poised at different potentials indicate a structural modification of the heme with the most negative potential during the first step of reduction. The correspondence between the redox sites as characterized by the EPR potentiometric titration and the hemes in the tridimensional structure is discussed.Cytochromes c3 (Mr 13000) are tetraheme proteins with low redox potentials found in sulfate-reducing bacteria. One of their main functions is to transfer electrons to their physiological partners, hydrogenase and ferredoxin. Each heme is covalently bound to the polypeptide chain and the iron atom is axially coordinated by two histidine side chains. The threedimensional structure has been established for two of them, Desuljovibrio desuljiuricans Norway cytochrome c3 (DdNc3) [l] and Desulfovibrio vulgaris Miyazaki cytochrome c3 (DvMc3) [2]. From the examination of the structure and the kinetic data, speculative models have been proposed for the intramolecular electron transfer pathways, mainly based on possible interactions between neighbouring redox sites [2, 51. The existence of such interactions is strongly suggested by the close packing of the heme groups within the protein and the presence of aromatic residues which can facilitate orbital overlaps. One may expect in such systems that the redox potential of each heme would depend upon the redox states of its neighbours. An example of a multisite protein, where effects of interactions on the redox equilibrium have been observed, is provided by cytochrome oxidase [7, 81. A formally similar situation is encountered in the study of the stoichiometric equilibria for the binding of a given ligand by a macromolecule with several binding sites [9, lo] been reported in Desuljovibrio gigas cytochrome c3 (Dgc3) from a redox study by NMR spectroscopy [12].The...