A Pulse‐Radiolysis Study of Cytochrome c₃

Abstract

1. Pulse-radiolysis experiments were performed in the presence of methyl viologen and cytochrome c3. After the pulse, methyl viologen radicals are formed and the kinetics of these radicals with cytochrome c3 are studied. The reaction between cytochrome c3 and methyl viologen radicals (MV') is diffusion controlled. The ionic strength dependence and the pH-dependence of this reaction were studied. From the ionic strength dependence (at pH 7.8) we found that the net charge of the fully oxidized cytochrome c3 molecule was Z = + 4.7 f 0.7.2. After the pulse an equilibrium is reached for the reaction of MV' with cytochrome c'3. From this equilibrium an apparent midpoint potential can be obtained. The apparent midpoint potential of this multihaem molecule was found to depend on the degree of reduction, E. With the help of the Nernst equation an empirical equation is obtained to describe this dependence of the midpoint potential: EO = -0.250-0.088~ (in V).3. An estimation is made of the energy of interaction between the haems due to electrostatic interactions (At; < 32 mV) and due to ionic strength effects (-12 mV < Ae < 26 mV). The results suggest that the redox properties of the individual haems in the cytochrome c3 molecule are dependent on the degree of reduction of the other haems in the molecule. 4. The reaction of cytochrome c3 with MV' or with ethanol radicals (EtOH') has been compared with the reactions of horse-heart cytochrome c and of metmyoglobin with the same radicals. The reaction of MV' or EtOH' with horse-heart cytochrome c is found to be diffusion controlled; the reactions with metmyoglobin on the other hand are most probably controlled by an activation energy.Cytochrome c3 from the sulphate-reducing bacterium Desulfovibrio vulgaris strain Hildenborough, NCIB 8303, is a well characterized protein [I -31 which contains four closely packed haem c groups. Its relative molecular mass is 13000 and it is easily soluble in water. The physiological role of cytochrome c3 is to act as an electron carrier between hydrogenase and ferredoxin. In the sulphate-reducing mode cytochrome c3 accepts electrons from hydrogenase and in the pyruvate-oxidating mode it donates electrons to hydrogenase [2,4]. One needs to know the midpoint potentials of the four haems in order to be able to describe these electron-transfer processes. It is generally accepted that the midpoint potentials of the four haems are located between -350mV and -250 mV [2,5-81. It is still not known whether the redox properties of the cytochrome c3 molecule should be described by the sum of four sites each of which has a fixed midpoint potential or by four redox sites whose midpoint potentials are not fixed but can vary depending on the interactions between the sites and the degree of reduction of the protein.EPR measurements of cytochrome c3 from D. vulgaris at 10 K have been analysed in terms of four fixed midpoint potentials: -284 mV, -310 mV, -319 mV, and -324 mV [5]. These values are microscopic midpoint potentials, i.e. they are characteristic valu...

Redox Mediators in Microbial Electrochemical Systems

“…That particularly matters for the cases using hydrophilic mediators to bridge the electron flux between suspended cells and the electrode. Kinetically, it has been shown that the intramolecular electron transfer rate constant between the microbial membrane protein hemes can reach 10 5 –10 9 s −1 , [75–77] and the intermolecular electron transfer rate constant between the heme and external electron acceptor is also on the level of 10 8 M −1 s −1 [78] . All these considerations above raise critical concerns of the transmembrane electron transfer (via artificial or self‐secreted electron carriers) as the potential rate‐limiting step for the MET pathways.…”