Thermodynamic and kinetic characterization of trihaem cytochrome c₃ from Desulfuromonas acetoxidans

Abstract

Trihaem cytochrome c 3 (also known as cytochrome c 551.5 and cytochrome c 7 ) is isolated from the periplasmic space of Desulfuromonas acetoxidans, a sulfur-reducing bacterium. Thermodynamic and kinetic data for the trihaem cytochrome c 3 are presented and discussed in the context of the possible physiological implications of its functional properties with respect to the natural habitat of D. acetoxidans, namely as a symbiont with green sulfur bacteria working as a mini-sulfuretum. The thermodynamic properties were determined through the fit of redox titration data, followed by NMR and visible spectroscopy, to a model of four functional centres that describes the network of cooperativities between the three haems and one protolytic centre. The kinetics of trihaem cytochrome c 3 reduction by sodium dithionite were studied using the stopped-flow technique and the data were fitted to a kinetic model that makes use of the thermodynamic properties to obtain the rate constants of the individual haems. This analysis indicates that the electrons enter the cytochrome mainly via haem I. The reduction potentials of the haems in this cytochrome show little variation with pH within the physiological range, and the kinetic studies show that the rates of reduction are also independent of pH in the range studied. Thus, although the trihaem cytochrome c 3 is readily reduced by hydrogenases from Desulfovibrio sp. and its haem core is similar to that of the homologous tetrahaem cytochromes c 3 , its physico-chemical properties are quite different, which suggests that these multihaem cytochromes with similar structures perform different functions.

Distance dependence of interactions between charged centres in proteins with common structural features

“…Detailed data on the distance dependence of redox interactions among the various centres in multiredox centre proteins are scarce, and current state of the art is limited to proteins with up to four redox centres. Interactions between charged centres were obtained from work on several multihaem cytochromes [19–22] [our manuscript in preparation]. In Fig.…”

Analysis of the electrochemistry of hemes with Ems spanning 800 mV

“… MCCE calculated vs. experimental redox potential (mV) 111–134. ( a ) All E m,calc vs. E m,expt (mV).…”

The role of intramolecular interactions in the functional control of multiheme cytochromes c

“…In contrast, PpcA and PpcD appear to be optimized to interact with specific redox partners involving e À /H + transfer via different mechanisms, where hemes I and IV are directly involved in the electronproton coupling. In the case of PpcA, electron-proton coupling occurs between oxidation stages 1 and 2, whereas in PpcD oxidation stages 0 and 2 are involved in a two-electron transfer step coupled to Squares illustrate data for the STC from the Shewanella genus [30,31]; Triangles illustrate data for the flavocytochromes c 3 from the Shewanella genus [32]; Diamonds illustrate data for the cytochrome c 7 /Ppc from Desulfuromonas and Geobacter genera [24,25]; the rectangle illustrates the range of the interactions reported for the cytochrome c 4 [33]; open circles illustrate data for the cytochromes c 3 from the Desulfovibrio and Desulfomicrobium genera [27][28][29]. Distances were measured between iron atoms from the protein structures with the following PDB codes: 1M1Q; 2K3V; 1QJD; 1D4D; 1HH5; 2LDO; 3BXU; 3H4N; 3H34; 1RWJ; 1WAD; 2CTH; 1UPD; 2BQ4; 2CY3; 1W7O; 1M70, using the program Pymol v0.99.…”