Scanning Probe Microscopy Techniques for the Study of Redox Proteins

Scanning probe microscopy (SPM) techniques provide a unique single molecule scale insight into the properties of redox proteins, which play key roles in biological electron-transfer (ET) chains. Here, we combine Electrochemical Scanning Tunneling Microscopy (ECSTM) and Atomic Force Microscopy (AFM) to study the charge-transport behavior and conformational stability of the photosynthetic redox protein plastocyanin (Pc), with and without copper cofactor, under varying pH conditions. Using a mutant Pc immobilized on Au(111), ECSTM current–distance (I–z) measurements reveal long-range charge transport beyond the classical tunneling regime. The distance-decay constant β decreases with increasing pH for both the holoprotein (Pcholo) and the copper-free apoprotein (Pcapo), Statistical analysis of the results yields lower β values (longer-extending currents) at higher pH both with Pcapo (plastocyanin protein without Cu) and Pcholo (plastocyanin protein with Cu). A similar trend with pH is also observed in experiments in Au (111) alone, but the β values here are higher (β > 7 nm-1, in a range compatible with electron tunneling between the ECSTM probe and Au (111) sample) than in Pc. These results support the observations reported in [1] for Pc-PSI and reveal the pH dependence of Pc ETp.
Complementary AFM single-molecule unfolding experiments show that the frequency of unfolding events and the maximum protein extension increase with pH, revealing higher structural lability at low proton concentration. Gold-coated AFM tips enable reproducible force spectroscopy signals with unfolding lengths up to ~30 nm, while the absence of significant events with silicon-nitride tips confirms specific Pc–Au interactions. These results demonstrate that pH-driven changes in Pc structural stability—particularly involving residues such as His37—correlate with the electrochemically observed reduction in conductivity. Together, ECSTM and AFM reveal how protonation state, copper coordination, and conformational flexibility jointly modulate the charge-transport properties of Pc, providing a comprehensive SPM-based framework to analyze redox proteins at the nanoscale.

[1] López-Ortiz, M.; Zamora, R. A.; Giannotti, M. I.; Gorostiza, P. The protein matrix of plastocyanin supports long-distance charge transport with photosystem I and the copper ion regulates its spatial span and conductance. ACS Nano 2023, 17, 20334 – 20344.