Genetically Encoded Photosensitizer Protein Reduces Iron-Sulfur Clusters of Radical SAM Enzymes

Radical S-adenosylmethionine (SAM) enzymes are a family of proteins with hundreds of thousands of members that all contain an essential [4Fe-4S] cluster. The [4Fe-4S] cluster needs to be reduced to cleave SAM and generate a 5'-deoxyadenosyl or 3-amino-3-carboxypropyl radical. Due to the low reduction potential of the [4Fe-4S] cluster, dithionite has been widely used in studies on the activity of radical SAM enzymes in vitro. Inspired by the efficient photoreduction of the [4Fe-4S] cluster in natural photosystem I, we applied the 27 kDa photosensitizer protein PSP2, which we reported previously, to reduce the [4Fe-4S] cluster of radical SAM enzymes. Herein, we demonstrate that light-activated PSP2 generates a PSP2 radical, which is capable of reducing the catalytic [4Fe-4S] cluster of the radical SAM enzyme BtrN. PSP2-reduced BtrN cleaves SAM to generate 5'-deoxyadenosine and converts the substrate 2-deoxy-scyllo-inosamine to 3-amino-2,3-dideoxy-scyllo-inosose. Remarkably, our study shows that PSP2 can reduce the auxiliary [4Fe-4S] cluster of BtrN that has the lowest reduction potential among the known radical SAM enzymes and cannot be reduced by dithionite. The reduction of this auxiliary cluster experimentally proves its function in product formation. In addition, we show that PSP2 can reduce the [4Fe-4S] cluster of Dph2, a noncanonical radical SAM enzyme involved in diphthamide biosynthesis, and help the modification of the substrate protein elongation factor 2. Therefore, PSP2 is a powerful photoreductant for radical SAM enzymes and a useful tool for studying clusters with low reduction potential.