The novel "hyper-reduced" form of protein-bound [3Fe-4S] clusters, which is two electron equivalents below the normal reduced form [3Fe-4S](0) and thus formally composed entirely of Fe(II) subsites, has been characterized by electrochemistry and by EPR, MCD, and UV/visible spectroscopy. The two-electron reduction of [3Fe-4S](0) has been studied for a range of proteins, in particular the 7Fe ferredoxins from Sulfolobus acidocaldarius, Desulfovibrio africanus, and Azotobacter vinelandii. In each case, the reaction is chemically reversible, the product is surprisingly inert, and the pH-dependent reduction potential is in the region of -700 mV vs SHE at pH 7, regardless of the identity of the protein. Protein film voltammetry of three different ferredoxins investigated in detail over a wide pH range shows that the novel species denoted as [3Fe-4S](2-) is formed by a cooperative two-electron reduction of [3Fe-4S](0) and there is a net uptake of three protons relative to the all-Fe(III) state [3Fe-4S](1+).