Modification of specific lysine residue of cytochrome c(3) with 2,4,6-trinitorobenzenesulfonic acid sodium salt dehydrate (TNBS) was carried out. By comparing the hydrogen evolution rate with modified cytochrome c(3) and native cytochrome c(3) catalyzed by hydrogenase, the role of lysine residue of cytochrome c(3) was discussed. The modified lysine residue was responsible for the intermolecular electron transfer from cytochrome c(3) to hydrogenase.