Attributes of the [4Fe4S] clusters in archaean species sulfolobus solfataricus Cas4 SSO0001

Abstract

Iron sulfur cluster is a ubiquitous protein cofactor that is reported to be essential to life. A wide range of protein families are reported to be iron sulfur coordinating, catalyzing reactions involving electron transfer and regulating gene expression. Emerging numbers of DNA-interacting enzymes were revealed to be iron sulfur cluster containing though the threat of reactive oxygen species generated by iron in close proximity with DNA was previously highlighted to be a concern. CRISPR-associated (Cas) proteins, the critical player in prokaryotic immunological defense against conjugative plasmids and phages, were well known to be nucleases able to digest DNA at dedicated sites given with guiding information from protospacer adjacent motif (PAM). Research of Cas4 presenting in Type I CRISPR-Cas systems proved the protein to be crucial in telling host spacer insertion polarity as hinted by PAM. Crystal structure of S. solfataricus Cas4 protein (Sso0001) was solved to contain [4Fe4S] which mutation of cluster coordinating cysteines resulted in structural instability.

Here, redox studies of [4Fe4S] within Sso0001 was explored on DNA-modified electrodes based on the principle of DNA charge transport (DNA CT). Cluster bulk electrolysis effect on Sso0001 exonuclease activity was examined so as to evaluate the relationship between cluster oxidative state and protein-DNA affinity. Fluorescein intensity of resolved denaturing gel bands suggested Sso0001 redox state could be a switch in controlling DNA binding efficiency, where the mutagenesis results of aromatic residue mutants (F184A, W186A and Y190A) proposed the indispensable role of W186 and Y190 in paving CT pathway between cluster and DNA in toroid space. Though the molecular basis of Cas4 was not fully addressed, this study had nonetheless explored the primary signalling function of cluster within Sso0001. S. solfataricus Cas4 bearing redox active [4Fe4S] cluster could be electrochemically characterized on sequence specific-DNA gold electrodes, thus visioning a sensitive and low-cost PAM screening method for other iron sulfur cluster containing Cas4 proteins.