The tetraheme cytochrome c(3) isolated from Desulfomicrobium baculatum (DSM 1743) (Dsmb) was cloned, and the sequence analysis showed that this cytochrome differs in just three amino acid residues from the cytochrome c(3) isolated from Desulfomicrobium norvegicum (Dsmn): (DsmnXXDsmb) Thr-37 --> Ser, Val-45 --> Ala, and Phe-88 --> Tyr. X-ray crystallography was used to determine the structure of cytochrome c(3) from Dsmb, showing that it is very similar to the published structure of cytochrome c(3) from Dsmn. A detailed thermodynamic and kinetic characterization of these two tetraheme cytochromes c(3) was performed by using NMR and visible spectroscopy. The results obtained show that the network of cooperativities between the redox and protonic centers is consistent with a synergetic process to stimulate the hydrogen uptake activity of hydrogenase. This is achieved by increasing the affinity of the cytochrome for protons through binding electrons and, reciprocally, by favoring a concerted two-electron transfer assisted by the binding of proton(s). The data were analyzed within the framework of the differences in the primary and tertiary structures of the two proteins, showing that residue 88, close to heme I, is the main cause for the differences in the microscopic thermodynamic parameters obtained for these two cytochromes c(3). This comparison reveals how replacement of a single amino acid can tune the functional properties of energy-transducing proteins, so that they can be optimized to suit the bioenergetic constraints of specific habitats.