Rubredoxins contain a mononuclear iron tetrahedrally coordinated by four cysteinyl sulfurs. We have studied the wild-type protein from Clostridium pasteurianum and two mutated forms, C9S and C42S, in the oxidized and reduced states, with Mössbauer, integer-spin EPR, and magnetic circular dichroism (MCD) spectroscopies. The Mössbauer spectra of the ferric C42S and C9S mutant forms yielded zero-field splittings, D = 1.2 cm(-1), that are about 40% smaller than the D-value of the wild-type protein. The 57Fe hyperfine coupling constants were found to be ca. 8% larger than those of the wild-type proteins. The present study also revealed that the ferric wild-type protein has delta=0.24+/-0.01 mm/s at 4.2 K rather than delta = 0.32 mm/s as reported in the literature. The Mössbauer spectra of both dithionite-reduced mutant proteins revealed the presence of two ferrous forms, A and B. These forms have isomer shifts delta = 0.79 mm/s at 4.2 K, consistent with tetrahedral Fe2+(Cys)3(O-R) coordination. The zero-field splittings of the two forms differ substantially; we found D = -7+/-1 cm(-1), E/D = 0.09 for form A and D = +6.2+/-1.3 cm(-1), E/D = 0.15 for form B. Form A exhibits a well-defined integer-spin EPR signal; from studies at X- and Q-band we obtained g(z) = 2.08+/-0.01, which is the first measured g-value for any ferrous rubredoxin. It is known from X-ray crystallographic studies that ferric C42S rubredoxin is coordinated by a serine oxygen. We achieved 75% reduction of C42S rubredoxin by irradiating an oxidized sample at 77 K with synchrotron X-rays; the radiolytic reduction produced exclusively form A, suggesting that this form represents a serine-bound Fe2+ site. Studies in different buffers in the pH 6-9 range showed that the A:B ratios, but not the spectral parameters of A and B, are buffer dependent, but no systematic variation of the ratio of the two forms with pH was observed. The presence of glycerol (30-50 % v/v) was found to favor the B form. Previous absorption and circular dichroism studies of reduced wild-type rubredoxin have suggested d-d bands at 7400, 6000, and 3700 cm(-1). Our low-temperature MCD measurements place the two high-energy transitions at ca. 5900 and 6300 cm(-1); a third d-d transition, if present, must occur with energy lower than 3300 cm(-1). The mutant proteins have d-d transitions at slightly lower energy, namely 5730, 6100 cm(-1) in form A and 5350, 6380 cm(-1) in form B.