The tetraheme cytochrome from Shewanella oneidensis MR-1 shows thermodynamic bias for functional specificity of the hemes

Abstract

Bacteria of the genus Shewanella contain an abundant small tetraheme cytochrome in their periplasm when growing anaerobically. Data collected for the protein isolated from S. oneidensis MR-1 and S. frigidimarina indicate differences in the order of oxidation of the hemes. A detailed thermodynamic characterization of the cytochrome from S. oneidensis MR-1 in the physiological pH range was performed, with data collected in the pH range 5.5-9.0 from NMR experiments using partially oxidized samples and from redox titrations followed by visible spectroscopy. These data allow the parsing of the redox and redox-protonation interactions that occur during the titration of hemes. The results show that electrostatic effects dominate the heme-heme interactions, in agreement with modest redox-linked structural modifications, and protonation has a considerable influence on the redox properties of the hemes in the physiological pH range. Theoretical calculations using the oxidized and reduced structures of this protein reveal that the bulk redox-Bohr effect arises from the aggregate fractional titration of several of the heme propionates. This detailed characterization of the thermodynamic properties of the cytochrome shows that only a few of the multiple microscopic redox states that the protein can access are significantly populated at physiological pH. On this basis a functional pathway for the redox activity of the small tetraheme cytochrome from S. oneidensis MR-1 is proposed, where reduction and protonation are thermodynamically coupled in the physiological range. The differences between the small tetraheme cytochromes from the two organisms are discussed in the context of their biological role.

Molecular Basis for Directional Electron Transfer

“…NMR data show that these conditions are met by both STCs studied in this work at the typical concentration used for stopped-flow experiments (10,11). For STC, the complete diagram of redox microstates involves 16 protonated and 16 deprotonated microstates (Fig.…”

“…Protein Sample-Tetraheme cytochromes were purified from the soluble fraction of Shewanella as previously described for SoSTC (10) and SfSTC (6). Stock solutions of SoSTC and SfSTC were degassed with cycles of vacuum and argon to remove dissolved oxygen.…”

“…This proposal was refined by considering that hemes I-III would feed electrons to heme IV on the basis of the thermodynamic characterization of SoSTC at high pH (9). With the full thermodynamic characterization in the physiological pH range, specific roles for the hemes were proposed, with heme I serving as the electron entry gate and heme IV as the electron exit (10). A similar situation was found for SfSTC, but the roles of the hemes I and IV are exchanged (11).…”

Functional Characterization of the FoxE Iron Oxidoreductase from the Photoferrotroph Rhodobacter ferrooxidans SW2

“…One-dimensional NMR spectra of partially reduced FoxE present no shifts of the LP signals toward the diamagnetic chemical shift position of the reduced state. This suggests that intramolecule electron transfer is slow in the NMR timescale (24). With a NOESY mixing time of 25 ms and using the relationship between distance and electron transfer rate (25), all the hemes in the oligomer are estimated to be more than 16 Å apart from each other.…”

“…This influence of pH (redox-Bohr effect) indicates that electron transfer is thermodynamically coupled to proton transfer in the physiological pH range. In other multiheme cytochromes the heme propionates are often identified as the major contributors to this effect (24). In environments with circumneutral pH, ferrous iron has many different forms with reduction potentials ranging from Ϫ200 mV to almost 400 mV (27).…”