BtrN is a radical SAM ( S-adenosyl- l-methionine) enzyme that catalyzes the oxidation of 2-deoxy- scyllo-inosamine (DOIA) into 3-amino-2,3-dideoxy- scyllo-inosose (amino-DOI) during the biosynthesis of 2-deoxystreptamine (DOS) in the butirosin producer Bacillus circulans. Recently, we have shown that BtrN catalyzes the transfer of a hydrogen atom at C-3 of DOIA to 5'-deoxyadenosine, and thus, the reaction was proposed to proceed through the hydrogen atom abstraction by the 5'-deoxyadenosyl radical. In this work, the BtrN reaction was analyzed by EPR spectroscopy. A sharp double triplet EPR signal was observed when the EPR spectrum of the enzyme reaction mixture was recorded at 50 K. The spin coupling with protons partially disappeared by reaction with [2,2- (2)H 2]DOIA, which unambiguously proved the observed signal to be a radical on C-3 of DOIA. On the other hand, the EPR spectrum of the [4Fe-4S] cluster of BtrN during the reaction showed a complex signal due to the presence of several species. Comparison of signals derived from a [4Fe-4S] center of BtrN incubated with various combinations of products (5'-deoxyadenosine, l-methionine, and amino-DOI) and substrates (SAM and DOIA) indicated that the EPR signals observed during the reaction were derived from free BtrN, a BtrN-SAM complex, and a BtrN-SAM-DOIA complex. Significant changes in the EPR signals upon binding of SAM and DOIA suggest the close interaction of both substrates with the [4Fe-4S] cluster.