Tumor suppressor protein 101F6, a gene product of the 3p21.3 (human) and 9F1 (mouse) chromosomal region, has recently been identified as a member of the cytochrome b561 (Cyt-b561) protein family by sequence homology. The His(6)-tagged recombinant mouse tumor suppressor Cyt-b561 protein (TSCytb) was recently expressed in yeast and purified, and the ascorbate reducibility was determined. TSCytb is auto-oxidizable and has two distinct heme b centers with redox potentials of approximately 40 and approximately 140 mV. Its split alpha-band in the dithionite-reduced spectrum at both 295 and 77 K is well resolved, and the separation between the two alpha-peaks is approximately 7 nm (approximately 222 cm(-1)). Singular value decomposition analysis of the split alpha-band in the ascorbate-reduced spectra revealed the presence of two major spectral components, each of them with split alpha-band but with different peak separations (6 and 8 nm). Similar minor differences in peak separation were obtained when the split alpha-bands in ascorbate-reduced difference spectra at low (<1 mM) and high (>10 mM) ascorbate concentrations were analysed. According to low-temperature electron paramagnetic resonance (EPR) spectroscopy, the two heme b centers are in the low-spin ferric state with maximum principal g values of 3.61 and 2.96, respectively. These values differ from the ones observed for other members of the Cyt-b561 family. According to resonance Raman spectroscopy, the porphyrin rings are in a relaxed state. The spectroscopic results are only partially in agreement with those obtained earlier for the native chromaffin granule Cyt-b561.