Single Molecule Force Spectroscopy Reveals That Two-coordinate Ferric Site Is Critical for The Folding of Holo-rubredoxin
Metalloproteins play important roles in a wide range of biological processes. The folding process of metalloproteins is complex due to the synergistic effects of the folding of their polypeptide chains and the incorporation of metal cofactors. The folding mechanism of the simplest iron-sulfur protein rubredoxin, which contains one ferric ion coordinated by four cysteinyl sulfurs, is revealed by optical tweezers for the first time. The folding of rubredoxin polypeptide chain is rapid and robust, while the reconstitution of the iron-sulfur center is greatly dependent upon the coordination state of the ferric ion on the unfolded polypeptide chain. If ferric ion is coordinated by two neighboring cysteine, rubredoxin can readily fold with the iron-sulfur center fully reconstituted. However, if ferric ion is only mono-coordinated, rubredoxin folds but the iron-sulfur center is not reconstituted. Our results suggested that folding of holo-rubredoxin follows a novel binding-folding-reconstitution mechanism, which is distinct from the folding mechanisms proposed for the folding of metalloprotein. Our study highlights the critical importance of the two-coordinate ferric site on the folding of holo-rubredoxin, which may have some important implications to our understanding of the folding mechanism of more complex metalloproteins in vivo.
- This article is part of the themed collection: 2020 Nanoscale HOT Article Collection