Study On Intramolecular Electron Transfer Mechanism Of The Rusticyanin

Rusticyanin is a member of type I copper protein, which considered as the majorcomponent in the iron respiratory electron transport chain of A. ferrooxidans. The ioncopper is coordinated in a distorted tetrahedral geometry with three strongly ligandsCys138, His85, His143and a relatively weaker ligand Met148. Both His85andCys138residues have been proved essential to binding with ion copper in Rusticyanin.The axial ligand of Met148have been extensively studied and it was revealed at someextent the distance between cooper atom and protein determined the level of the redoxpotential of Rusticyanin, but this is not the most important factor. Meanwhile, to dateonly a few studies have been done toward residue His143, but it is likely that residueHis143plays a key residue as a “redox switch”, nevertheless no report found.In this research, the gene of rusticyanin was cloned and constructed the plasmids,then expressed in Escherichia coli competent cells, and later purified using one-stepaffinity chromatography. The absorption spectra study results confirmed that therusticyanin have reaction with IRO. These results confirmed that the electron transferbetween rusticyanin and IRO is conducted in the iron respiratory electron transportchain of A. ferrooxidans.We furthermore constructed the mutant expression plasmids of residue His143using site-directed mutagenesis. Mutant proteins were expressed in E.coli and purifiedwith a nickel metal affinity column. The absorption spectra study showed that His143is a key residue for the high reduction potential of Rusticyanin. Removal of theimidazole group of His143residue resulted in copper reduction and loss ofactivity.We found recovery of activity when the mutant protein143Gly was incubatedin imidazole solution. Molecular structure modeling showed that mutation ofimidazole group caused exposure of copper ions to environment solution, the solventoutside can easily go inside and caused the copper ions structure collapsed, thuselectron transferred activity disrupted. Hence, His143residue is a key component ofelectron transfer in Rusticyanin.