A new non-heme iron protein from the periplasmic fraction of Desulfovibrio vulgaris (Hil-denbourough NCIB 8303) has been purified to homogeneity, and its amino acid composition, molecular weight, redox potential, iron content, and optical, EPR, and Mossbauer spectroscopic properties have been determined. This new protein is composed of two identical subunits with subunit molecular weight of 21 900 and contains four iron atoms per molecule. The as-purified oxidized protein exhibits an optical spectrum with absorption maxima at 492, 365, and 280 nm, and its EPR spectrum shows resonances at g = 4,3 and 9.4, characteristic of oxidized rubredoxin. The Mössbauer data indicate the presence of approximately equal amounts of two types of iron; we named them the Rd-like and the Hr-like iron due to their similarity to the iron centers of rubredoxins (Rds) and hemerythrins (Hrs), respectively. For the Rd-like iron, the measured fine and hyperfine parameters (D = 1.5 cm-1, E/D = 0.26, ΔEQ= -0.55 mm/s, δ= 0.27 mm/s, Axx/gnβn= -16.5 T, Ayy/gnβn= -15.6 T, and Azz/gnβn= -17.0 T) are almost identical with those obtained for the rubredoxin from Clostridium pasteurianum. Redox-titration studies monitored by EPR, however, showed that these Rd-like centers have a midpoint redox potential of +230 ± 10 mV, approximately 250 mV more positive than those reported for rubredoxins. Another unusual feature of this protein is the presence of the Hr-like iron atoms. At 4.2 K, the Mossbauer spectrum of these Hr-like iron atoms is a quadrupole doublet with parameters (ΔEQ= 1.47 ± 0.05 mm/s and δ= 0.52 ± 0.03 mm/s) typical of high-spin ferric ions (S =5/2) with oxygenous and/or nitrogenous ligands. Mossbauer spectra recorded with strong magnetic fields up to 8 T indicate that these Hr-like ions are in a diamagnetic (S = 0) environment. Consequently, these two Hr-like iron atoms must be strongly antiferromagnetically coupled to form a diamagnetic binuclear center. Similar Mossbauer properties have been observed for the binuclear iron center in the methemerythrin. As for the reduced hemerythrin, the reduced Hr-like center in this new protein exhibits a single quadrupole doublet with parameters (ΔEQ= 3.14 ± 0.04 mm/s and δ = 1.30 =0.03 mm/s) typical of high-spin ferrous ions. Also, the EPR spectrum of the native protein exhibits a very weak signal at g = 1.98, 1.76, and 1.57, very similar to those signals reported for the binuclear iron centers in the semimet forms of hemerythrin. In conclusion, our spectroscopic data indicate unambiguously that this protein contains two rubredoxin-like FeS4centers and a hemerythrin-like binuclear iron cluster. Due to this unusual combination of prosthetic groups, this new protein is named rubrerythrin. The physiological function of rubrerythrin is presently unknown and is under investigation.