Ferrous oxygenated hemoglobins (Hb(2+)O2) autoxidize to ferric Hb(3+), but Hb(3+) is reduced to Hb(2+) by enzymatic and non-enzymatic mechanisms. We characterized the interaction between the soybean ferric leghemoglobin reductase 2 (FLbR2) and ferric rice non-symbiotic Hb1 (Hb1(3+)). Spectroscopic analysis showed that FLbR2 reduces Hb1(3+). Analysis by tryptophan fluorescence quenching showed that FLbR2 interacts with Hb1(3+), however the use of ITC and IEF techniques revealed that this interaction is weak. In silico modeling showed that predicted FLbR2 and native Hb1(3+) interact at the FAD-binding domain of FLbR2 and the CD-loop and helix F of Hb1(3+).