Cgr2 is sufficient for digoxin reduction and requires FAD and [4Fe-4S] cluster(s) for activity. ( A ) Whole cell assays using R. erythropoli ). ( B ) Annotation and amino acid numbering of Cgr2, including the predicted Tat secretion signal and three conserved flavin-binding motifs from the glutathione reductase family (X = any amino acid; h = hydrophobic residue). ( C ). FAD = flavin adenine dinucleotide; FMN = flavin mononucleotide. ( D ) Ultraviolet-visible (UV-Vis) absorption spectra of Cgr2 revealed an oxygen-sensitive peak centered around 400 nm that increased upon [Fe-S] cluster reconstitution, supporting the presence of [4Fe-4S] clusters in Cgr2. ( E ) Electron paramagnetic resonance (EPR) spectra of sodium dithionite-reduced Cgr2 reconstituted with iron ammonium sulfate hexahydrate ((NH 4 ) 2 Fe(SO 4 ) 2 ·6H 2 0) and sodium sulfide (Na 2 S·9H 2 0). G-values and decreased EPR signal intensity at higher temperatures (10 – 40 K) indicated the presence of low potential [4Fe-4S] 1+ clusters. Experimental conditions were microwave frequency 9.38 GHz, microwave power 0.2 mW, modulation amplitude 0.6 mT, and receiver gain 40 dB.
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