Quick and Spontaneous Transformation between [3Fe-4S] and [4Fe-4S] Iron-Sulfur Clusters in the tRNA-Thiolation Enzyme TtuA

• Published in: International journal of molecular sciences Vol. 24; no. 1; p. 833
• Main Authors: Ishizaka, Masato, Chen, Minghao, Narai, Shun, Tanaka, Yoshikazu, Ose, Toyoyuki, Horitani, Masaki, Yao, Min
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI 03.01.2023; MDPI AG
• Subjects: 2-thiouridine biosynthesis; EPR spectroscopy; iron–sulfur cluster; sulfurtransferase; tRNA sulfur modification; tRNA-thiolation; tRNA sulfur modification; tRNA-thiolation; iron–sulfur cluster; sulfurtransferase; redox biochemistry; EPR spectroscopy; 2-thiouridine biosynthesis
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms24010833

Iron-sulfur (Fe-S) clusters are essential cofactors for enzyme activity. These Fe-S clusters are present in structurally diverse forms, including [4Fe-4S] and [3Fe-4S]. Type-identification of the Fe-S cluster is indispensable in understanding the catalytic mechanism of enzymes. However, identifying [4Fe-4S] and [3Fe-4S] clusters in particular is challenging because of their rapid transformation in response to oxidation-reduction events. In this study, we focused on the relationship between the Fe-S cluster type and the catalytic activity of a tRNA-thiolation enzyme (TtuA). We reconstituted [4Fe-4S]-TtuA, prepared [3Fe-4S]-TtuA by oxidizing [4Fe-4S]-TtuA under strictly anaerobic conditions, and then observed changes in the Fe-S clusters in the samples and the enzymatic activity in the time-course experiments. Electron paramagnetic resonance analysis revealed that [3Fe-4S]-TtuA spontaneously transforms into [4Fe-4S]-TtuA in minutes to one hour without an additional free Fe source in the solution. Although the TtuA immediately after oxidation of [4Fe-4S]-TtuA was inactive [3Fe-4S]-TtuA, its activity recovered to a significant level compared to [4Fe-4S]-TtuA after one hour, corresponding to an increase of [4Fe-4S]-TtuA in the solution. Our findings reveal that [3Fe-4S]-TtuA is highly inactive and unstable. Moreover, time-course analysis of structural changes and activity under strictly anaerobic conditions further unraveled the Fe-S cluster type used by the tRNA-thiolation enzyme.