Spectral characterization of the recombinant mouse tumor suppressor 101F6 protein.
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.
Bérczi
Alajos
A
Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, Temesvári krt. 62, P.O. Box 521, 6701, Szeged, Hungary. berczi@brc.hu
Desmet
Filip
F
Van Doorslaer
Sabine
S
Asard
Han
H
D000595:Amino Acid Sequence; D000818:Animals; D003573:Cytochrome b Group / Q000737:chemistry* / Q000235:genetics; D004227:Dithionite; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D006418:Heme / Q000737:chemistry; D008565:Membrane Proteins; D051379:Mice; D008823:Micelles; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012440:Saccharomyces; D017386:Sequence Homology, Amino Acid; D013057:Spectrum Analysis / Q000379:methods; D013059:Spectrum Analysis, Raman; D013696:Temperature; D025521:Tumor Suppressor Proteins / Q000737:chemistry* / Q000235:genetics; D014466:Ultraviolet Rays
C529422:Cyb561d2 protein, mouse; D003573:Cytochrome b Group; D008565:Membrane Proteins; D008823:Micelles; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; D004227:Dithionite; D006418:Heme