Biological Semiconductors: Structural Control of Heme Redox Potentials in PpcA, a 3-Heme Cytochrome

Abstract

In an effort to tune and optimize relative bandgap potentials found in biological nanowires that form from multiheme domains of members of the cytochrome c7 family, we developed and extensively characterized 12 point mutations of PpcA, a 3-heme member of the cytochrome c7 family native to Geobacter sulfurreducens. These mutations were engineered to influence the redox potential (Em) of the middle heme (heme III) in PpcA by using four different strategies: performing charge reversal mutations, decreasing solvent access to the heme plane with bulky residues, altering the native bis-histidine axial ligation of the heme, and by attempting to form hydrogen bonds with the propionates of the heme. The latter strategy is expected not only to increase Em but also to introduce a redox Bohr effect. Out of 12 mutants, 11 were successfully expressed in E.coli and show thermal stability in temperature-dependent CD experiments comparable to the wild-type protein (Tm > 90°C). HPLC-ESI-MS was used to confirm both the purity and the mass of the expressed mutants. Small-angle X-ray scattering confirmed that the mutant proteins formed the expected compact globular structures. In addition to ongoing attempts to obtain protein crystals for X-ray crystallography, we performed extensive all-atom molecular dynamics simulations for all mutants. Formation of propionate hydrogen bonds is supported by simulations for A19R and A23R, while increased solvent access is supported for A19I, A19R, A23R, H20M, and K60E. UV/Vis and NMR redox titrations were performed in order to measure the effect of the mutations on the electrochemical properties of all 3 hemes and to understand the underlying principles and viable approaches in tuning relative heme redox potentials. Successful development of this project may lead to biological semiconductors with much smaller footprints and selectively tunable bandgap properties.