Kinetic, electrochemical and spectral characterization of bacterial and archaeal rusticyanins; unexpected stability issues and consequences for applications in biotechnology

Motivated by the ambition to establish an enzyme-driven bioleaching pathway for copper extraction, properties of the Type-1 copper protein rusticyanin from Acidithiobacillus ferrooxidans (AfR) were compared with those from an ancestral form of this enzyme (N0) and an archaeal enzyme identified in Ferroplasma acidiphilum (FaR). While both N0 and FaR show redox potentials similar to that of AfR their electron transport rates were significantly slower. The lack of a correlation between the redox potentials and electron transfer rates indicates that AfR and its associated electron transfer chain evolved to specifically facilitate the efficient conversion of the energy of iron oxidation to ATP formation. In F. acidiphilum this pathway is not as efficient unless it is up-regulated by an as of yet unknown mechanism. In addition, while the electrochemical properties of AfR were consistent with previous data, previously unreported behavior was found leading to a form that is associated with a partially unfolded form of the protein. The cyclic voltammetry (CV) response of AfR immobilized onto an electrode showed limited stability, which may be connected to the presence of the partially unfolded state of this protein. Insights gained in this study may thus inform the engineering of optimized rusticyanin variants for bioleaching processes as well as enzyme-catalyzed solubilization of copper-containing ores such as chalcopyrite. Rusticyanin (AfR) plays a central role in the electron transfer chain that links iron oxidation to the energy metabolism. It may also be a suitable catalyst to promote the cell-free iron solubilization from complex orebodies (e.g., chalcopyrite). [Display omitted] •Bio-based metal extraction facilitate the decarbonisation of the mining industry.•Rusticyanin is a protein in an electron transfer chain that underpins bioleaching.•Rusticyanin may also facilitate the cell-free liberation of iron from ore bodies.•Immobilized rusticyanin is not very stable due to structural flexibility.•Restricting this flexibility may enhance its suitability for mining applications.