European Biophysics Journal

, Volume 39, Issue 8, pp 1129–1142 | Cite as

Spectral characterization of the recombinant mouse tumor suppressor 101F6 protein

  • Alajos Bérczi
  • Filip Desmet
  • Sabine Van Doorslaer
  • Han Asard
Original Paper

Abstract

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 His6-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 ~40 and ~140 mV. Its split α-band in the dithionite-reduced spectrum at both 295 and 77 K is well resolved, and the separation between the two α-peaks is ~7 nm (~222 cm−1). Singular value decomposition analysis of the split α-band in the ascorbate-reduced spectra revealed the presence of two major spectral components, each of them with split α-band but with different peak separations (6 and 8 nm). Similar minor differences in peak separation were obtained when the split α-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.

Keywords

Ascorbate Auto-oxidation Cyt-b561 protein EPR Raman UV–VIS 101F6 protein 

Notes

Acknowledgments

The authors thank Drs. Balázs Szalontai and Csaba Bagyinka (Institute of Biophysics, BRC, Szeged, Hungary) for their help in spectrum analysis. This work was supported by grants from the University of Antwerp (to H.A.). F.D. thanks the BOF-UA-TOP fund for PhD funding.

References

  1. Apps DK, Pryde JG, Phillips JH (1980) Cytochrome b561 is identical with chromomembrin B, a major polypeptide of chromaffin granule membranes. Neuroscience 5:2279–2287CrossRefPubMedGoogle Scholar
  2. Apps DK, Boisclair MD, Gavine FS, Pettigrew GW (1984) Unusual redox behaviour of cytochrome b-561 from bovine chromaffin granule membranes. Biochim Biophys Acta 764:8–16CrossRefPubMedGoogle Scholar
  3. Asada A, Kusakawa T, Orii H, Agata K, Watanabe K, Tsubaki M (2002) Planarian cytochrome b561: conservation of a six trans-membrane structure and localization along the central and peripheral nervous system. J Biochem 131:175–182PubMedGoogle Scholar
  4. Asard H, Kapila J, Verelst W, Bérczi A (2001) Higher-plant plasma membrane cytochrome b561: a protein in search of a function. Protoplasma 217:77–93CrossRefPubMedGoogle Scholar
  5. Babcock GT, Widger WR, Cramer WA, Oertling WA, Metz JG (1985) Axial ligands of chloroplast cytochrome b-559: identification and requirement for a heme-cross-linked polypeptide structure. Biochemistry 24:3638–3645CrossRefPubMedGoogle Scholar
  6. Baker PD, Nerou EP, Cheesman MR, Thomson AJ, de Oliveira P, Hill HAO (1996) Bis-Methionine ligation to heme iron in mutants of cytochrome b. 1. Spectroscopic and electrochemical characterization of the electronic properties. Biochemistry 35:13618–13626CrossRefGoogle Scholar
  7. Bashtovyy D, Bérczi A, Asard H, Páli T (2003) Structure prediction for di-heme cytochrome b561 protein family. Protoplasma 221:31–40CrossRefPubMedGoogle Scholar
  8. Bérczi A, Asard H (2008) Expression and purification of the recombinant mouse tumor suppressor cytochrome b561 protein. Acta Biol Szeged 52:257–265Google Scholar
  9. Bérczi A, Su D, Lakshminarasimhan M, Vargas A, Asard H (2005) Heterologous expression and site-directed mutagenesis of an ascorbate-reducible cytochrome b561. Arch Biochem Biophys 443:82–92CrossRefPubMedGoogle Scholar
  10. Bérczi A, Su D, Asard H (2007) An Arabidopsis cytochrome b561 with trans-membrane ferrireductase capability. FEBS Lett 581:1505–1508CrossRefPubMedGoogle Scholar
  11. Blumberg WE, Peisach J (1971) A unified theory for low-spin forms of all ferric heme proteins as studied by EPR. In: Chance B, Yonetani T, Mildvan AS (eds) Probes of structure and function of macromolecules and membranes. Probes of enzymes and hemoproteins, vol 2. Academic Press, New York, pp 215–229Google Scholar
  12. Bois-Poltoratsky R, Ehrenberg A (1967) Magnetic and spectrophotometric investigations of cytochrome b 5. Eur J Biochem 2:361–365CrossRefPubMedGoogle Scholar
  13. Branca RMM, Bodó G, Várkonyi Z, Debreczeny M, Ősz J, Bagyinka C(2007) Oxygen and temperature-dependent structural and redox changes in a novel cytochrome c 4 from the purple sulfur photosynthetic bacterium Thiocapsa roseopersicina. Arch Biochem Biophys 467:174–184CrossRefPubMedGoogle Scholar
  14. Cerda-Colon JF, Silfa E, Lopez-Garriga J (1998) CO-dependent activity controlling mechanism of heme-containing CO-sensor protein, NPAS2. J Am Chem Soc 120:9312–9317CrossRefGoogle Scholar
  15. Cheesman MR, Thomson AJ, Greenwood C, Moore GR, Kadir F (1990) Bis-methionine axial ligation of haem in bacterioferritin from Pseudomonas aeruginosa. Nature 346:771–773CrossRefPubMedGoogle Scholar
  16. Cheesman MR, Kadir FHA, Al-Basseet J, Al-Massad F, Farrar J, Greenwood C, Thomson AJ, Moore GF (1992) E.P.R. and magnetic circular dichroism spectroscopic characterization of bacterioferritin from Pseudomonas aeruginosa and Azotobacter vinelandii. Biochem J 286:361–367PubMedGoogle Scholar
  17. Dewilde S, Ebner B, Vinck E, Gilany K, Hankeln T, Burmester T, Kreiling J, Reinisch C, Vanfleteren JR, Kiger L, Marden MC, Hundahl C, Fago A, Van Doorslaer S, Moens L (2006) The nerve hemoglobin of the bivalve mollusc Spisula solidissima: molecular cloning, ligand binding studies, and phylogenetic analysis. J Biol Chem 281:5364–5372CrossRefPubMedGoogle Scholar
  18. Flatmark T, Grønberg M (1981) Cytochrome b-561 of the bovine adrenal chromaffin granules. Molecular weight and hydrodynamic properties in micellar solutions of Triton X-100. Biochem Biophys Res Commun 99:292–301CrossRefPubMedGoogle Scholar
  19. Flatmark T, Terland O (1971) Cytochrome b 561 of the bovine adrenal chromaffin granules. A high potential b-type cytochrome. Biochim Biophys Acta 253:487–491CrossRefPubMedGoogle Scholar
  20. Fleming PJ, Kent UM (1991) Cytochrome b561, ascorbic acid, and transmembrane electron transfer. Am J Clin Nutr 54:1173S–1178SPubMedGoogle Scholar
  21. Griesen D, Su D, Bérczi A, Asard H (2004) Localization of an ascorbate-reducible cytochrome b561 in the plant tonoplast. Plant Physiol 134:726–734CrossRefPubMedGoogle Scholar
  22. Hagihara B, Oshino R, Iizuka T (1974) Studies on low temperature spectra of respiratory pigments. II. Spectra of cytochromes in respiratory systems between liquid helium and room temperatures. J Biochem 75:45–51PubMedGoogle Scholar
  23. Henry ER, Hofrichter J (1992) Singular value decomposition: application to analysis of experimental data. Meth Enzymol 210:129–193CrossRefGoogle Scholar
  24. Hu S, Morris IK, Singh JP, Smith KM, Spiro TG (1993) Complete assignment of cytochrome c resonance Raman spectra via enzymatic reconstitution with isotopically labeled hemes. J Am Chem Soc 115:12446–12458CrossRefGoogle Scholar
  25. Hu S, Smith KM, Spiro TG (1996) Assignment of protoheme resonance Raman spectrum by heme labeling in myoglobin. J Am Chem Soc 118:12638–12646CrossRefGoogle Scholar
  26. Hurst B, Loehr TM, Curnutte JT, Rosen H (1991) Resonance Raman and electron paramagnetic resonance structural investigations of neutrophil cytochrome b558. J Biol Chem 266:1627–1634PubMedGoogle Scholar
  27. Ikeda M, Iizuka T, Takao H, Hagihara B (1974) Studies on heme environment of oxidized cytochrome b 5. Biochim Biophys Acta 226:15–24Google Scholar
  28. Jahn HA, Teller E (1937) Stability of polyatomic molecules in degenerate electronic states. I. Orbital degeneracy. Proc R Soc Lond Ser A 161:220–235CrossRefGoogle Scholar
  29. Ji L, Nizhizaki M, Gao B, Burbee D, Kondo M, Kamibayashi C, Xu K, Yen N (2002) Expression of several genes in the human chromosome 3p21.3 homozygous deletion region by an adenovirus vector results in tumor suppressor activities in vitro and in vivo. Cancer Res 62:2715–2720PubMedGoogle Scholar
  30. Kamensky YA, Palmer G (2001) Chromaffin granule membranes contain at least three heme centers: direct evidence from EPR and absorption spectroscopy. FEBS Lett 491:119–122CrossRefPubMedGoogle Scholar
  31. Kamensky Y, Liu W, Tsai A-L, Kulmacz RJ, Plamer G (2007) Axial ligation and stoichiometry of heme centers in adrenal cytochrome b561. Biochemistry 46:8647–8658CrossRefPubMedGoogle Scholar
  32. Kelley PM, Njus D (1986) Cytochrome b561 spectral changes associated with electron transfer in chromaffin-vesicle ghosts. J Biol Chem 261:6429–6432PubMedGoogle Scholar
  33. Kent UM, Fleming PJ (1987) Purified cytochrome b 561 catalyzes transmembrane electron transfer for dopamine β-hydroxylase and peptidyl glycine α-amidating monooxygenase activities in reconstituted systems. J Biol Chem 262:8174–8178PubMedGoogle Scholar
  34. La Mar GN, Toi H, Krishnamoorthi R (1984) Proton NMR investigation of the rate and mechanism of heme rotation in sperm whale myoglobin: evidence for intramolecular reorientation about a heme two-fold axis. J Am Chem Soc 106:6395–6401CrossRefGoogle Scholar
  35. Leenstra WR (1979) The triplet state of porphyrins. Ph.D. Thesis, University of Washington, Seattle, WA, USAGoogle Scholar
  36. Lerman MI, Minna JD (2000) The 630-kb lung cancer homozygous deletion region on human chromosome 3p21.3: identification and evaluation of the resident candidate tumor suppressor genes. Cancer Res 60:6116–6133PubMedGoogle Scholar
  37. Liu W, Kamensky Y, Kakkar R, Foley E, Kulmacz RJ, Palmer G (2005) Purification and characterization of bovine adrenal cytochrome b561 expressed in insect and yeast cell systems. Protein Expr Purif 40:429–439CrossRefPubMedGoogle Scholar
  38. Liu W, Rogge CE, Kamensky Y, Tsai A-L, Kulmacz RJ (2007) Development of a bacterial system for high yield expression of fully functional adrenal cytochrome b561. Protein Expr Purif 56:145–152CrossRefPubMedGoogle Scholar
  39. Liu W, Rogge CE, da Silva GFZ, Shinkarev VP, Tsai A-L, Kamensky Y, Palmer G, Kulmacz RJ (2008) His92 and His110 selectively affect different heme centers of adrenal cytochrome b561. Biochim Biophys Acta 1777:1218–1228CrossRefPubMedGoogle Scholar
  40. Lou BS, Snyder JK, Marshall P, Wang JS, Wu G, Kulmacz RJ, Tsai AL, Wang J (2000) Resonance Raman studies indicate a unique heme active site in prostaglandin H synthase. Biochemistry 39:12424–12434CrossRefPubMedGoogle Scholar
  41. Markwell MAK, Haas SB, Bieber LL, Tolbert NE (1978) A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem 87:206–210CrossRefPubMedGoogle Scholar
  42. McKie AT, Barrow D, Latunde-Dada GO, Rolfs A, Sager G, Mudaly E, Mudaly M, Richardson C, Barlow D, Bomford A, Peters RJ, Raja KB, Shirali S, Hediger MA, Farzaneh F, Simpson RJ (2001) As iron-regulated ferric reductase associated with the absorption of dietary iron. Science 291:1755–1759CrossRefPubMedGoogle Scholar
  43. McKnight J, Cheesman MR, Reed CA, Orosz R, Thomson AJ (1991) Comparative study of the optical and magnetic circular dichroism spectra of bis-thioether and -imidazole complexes of iron(III) tetraphenyl and octaethyl-porphyrin. Models of haem coordination in bacterioferritins. J Chem Soc Dalton Trans 1991(8):1887–1894Google Scholar
  44. Mizutani A, Sanuki R, Kakimoto K, Kojo S, Taketani S (2007) Involvement of 101F6, a homologue of cytochrome b561, in the reduction of ferric ions. J Biochem 142:699–705CrossRefPubMedGoogle Scholar
  45. Njus D, Kelley PM (1993) The secretory-vesicle ascorbate-regenerating system: a chain of concerted H+/e(−)-transfer reactions. Biochim Biophys Acta 1144:235–248CrossRefPubMedGoogle Scholar
  46. Ohtani O, Iwamaru A, Deng W, Ueda K, Wu G, Jayachandran G, Kondo S, Atkinson EN, Minna JD, Roth JA, Ji L (2007) Tumor suppressor 101F6 and ASC synergistically and selectively inhibit non–small cell lung cancer growth by caspase-independent apoptosis and autophagy. Cancer Res 67:6293–6303CrossRefPubMedGoogle Scholar
  47. Pearson RG (1975) Concerning Jahn-Teller effects. Proc Natl Acad Sci USA 72:2104–2106CrossRefPubMedGoogle Scholar
  48. Peisach J (1998) EPR of metalloproteins: truth tables revisited. In: Eaton GR, Eaton SS, Salikov K (eds) Foundations of modern EPR, 1st edn. World Scientific Publishing, Singapore, pp 346–360Google Scholar
  49. Peisach J, Blumberg WE (1974) Structural implications derived from the analysis of electron paramagnetic resonance spectra of natural and artificial copper proteins. Arch Biochem Biophys 165:691–708CrossRefPubMedGoogle Scholar
  50. Ponting CP (2001) Domain homologues of dopamine β-hydroxylase and ferric reductase: roles for iron metabolism in neurodegenerative disorders. Human Mol Gen 10:1853–1858CrossRefGoogle Scholar
  51. Quinn R, Valentine JS, Byrn MP, Strouse CE (1987) Electronic structure of low-spin ferric porphyrins: a single-crystal EPR and structural investigation of the influence of axial ligand orientation and the effects of pseudo-Jahn-Teller distortion. J Am Chem Soc 109:3301–3308CrossRefGoogle Scholar
  52. Ran Y, Zhu H, Liu M, Fabian M, Olson JS, Aranda R IV, Phillips GN, Dooley DM, Lei B (2007) Bis methionine ligation to heme iron in the streptococcal cell surface protein Shp facilitates rapid hemin transfer to HtsA of the HtsABC transporter. J Biol Chem 282:31380–31388CrossRefPubMedGoogle Scholar
  53. Recuenco MC, Fujito M, Rahman M, Sakamoto Y, Takeuchi F, Tsubaki M (2009) Functional expression and characterization of human 101F6 protein, a homologue of cytochrome b561 and a candidate tumor suppressor gene product. Biofactors 34:219–230CrossRefPubMedGoogle Scholar
  54. Reddy KS, Angiolillo PJ, Wright WW, Laberge M, Vanderkooi JM (1996) Spectral splitting in the α(Q0,0) absorption band of ferrous cytochrome c and other heme proteins. Biochemistry 35:12820–12830CrossRefPubMedGoogle Scholar
  55. Shifman JM, Gibney BR, Sharp RE, Dutton PL (2000) Heme redox potential control in de novo designed four-α-helix bundle proteins. Biochemistry 39:14813–14821CrossRefPubMedGoogle Scholar
  56. Shrager RI (1986) Chemical transitions measured by spectra and resolved using singular value decomposition. Chem Intell Lab Syst 1:59–70CrossRefGoogle Scholar
  57. Stoll S, Schweiger A (2006) EasySpin, a comprehensive software package for spectral simulation and analysis in EPR. J Magn Reson 178:42–55CrossRefPubMedGoogle Scholar
  58. Su D, Asard H (2006) Three mammalian cytochrome b 561 are ascorbate dependent ferrireductases. FEBS J 273:3722–3734CrossRefPubMedGoogle Scholar
  59. Takeuchi F, Kobayashi K, Tagawa S, Tsubaki M (2001) Ascorbate inhibits the carbethoxylation of two histidyl and one tyrosyl residues indispensable for the transmembrane electron transfer reaction of cytochrome b561. Biochemistry 40:4067–4076CrossRefPubMedGoogle Scholar
  60. Takeuchi F, Hori H, Obayashi E, Shiro Y, Tsubaki M (2004) Properties of two distinct centers of cytochrome b561 from bovine chromaffin vesicles studied by EPR, resonance Raman, and ascorbate reduction assay. J Biochem 135:53–64CrossRefPubMedGoogle Scholar
  61. Taylor CPS (1977) The EPR of low spin heme complexes. Relation of the t2g hole model to the directional properties of the g-tensor, and a new method for calculating the ligand field parameters. Biochim Biophys Acta 491:137–149PubMedGoogle Scholar
  62. Teixeira M, Campos AP, Aguiar AP, Costa HS, Santos H, Turner DL, Xavier AV (1993) Pitfalls in assigning heme axial coordination by EPR. C-type cytochromes with atypical Met-His ligation. FEBS Lett 317:233–236CrossRefPubMedGoogle Scholar
  63. Terekhov SN, Kruglik SG (1995) Photoreduction of ferric-tetraphenylporphyrin in oxygen-containing solvents revealed by resonance Raman and absorption spectroscopy. Chem Phys Lett 245:268–272CrossRefGoogle Scholar
  64. Tsubaki M, Nakayama M, Okuyam E, Ichikawa Y, Hori H (1997) Existence of two heme B centers in cytochrome b561 from bovine adrenal chromaffin vesicles as revealed by a new purification procedure and EPR spectroscopy. J Biol Chem 272:23206–23210CrossRefPubMedGoogle Scholar
  65. Tsubaki M, Takeuchi F, Nakanashi N (2005) Cytochrome b561 protein family: expanding roles and versatile transmembrane electron transfer abilities as predicted by a new classification system and protein sequence motif analyses. Biochim Biophys Acta 1753:174–190PubMedGoogle Scholar
  66. Tusnády GE, Simon I (1998) Principles governing amino acid composition of integral membrane proteins: application to topology prediction. J Mol Biol 283:489–506CrossRefPubMedGoogle Scholar
  67. Tusnády GE, Simon I (2001) The HMMTOP transmembrane topology prediction server. Bioinformatics 17:849–850CrossRefPubMedGoogle Scholar
  68. Uchida T, Sato E, Sato A, Sagami I, Shimizu T, Kitagawa T (2005) CO-dependent activity-controlling mechanism of heme-containing CO-sensor protein, neuronal PAS domain protein 2. J Biol Chem 280:21358–21368CrossRefPubMedGoogle Scholar
  69. Verelst W, Asard H (2003) A phylogenetic study of cytochrome b561 proteins. Genome Biol 4:R38CrossRefPubMedGoogle Scholar
  70. Wagner GC, Kassner RJ (1975) Spectroscopic properties of low-spin ferrous heme complexes and heme proteins at 77°K. Biochem Biophys Res Commun 63:385–391CrossRefPubMedGoogle Scholar
  71. Wakefield LM, Cass AEG, Radda GK (1984) Isolation of a membrane protein by chromatofocusing: Cytochrome b-561 of the adrenal chromaffin granule. J Biochem Biophys Methods 9:331–341CrossRefPubMedGoogle Scholar
  72. Walker FA (1999) Magnetic spectroscopic (EPR, ESEEM, Mössbauer, MCD and NMR) studies of low-spin ferriheme centers and their corresponding heme proteins. Coord Chem Rev 185–186:471–534CrossRefGoogle Scholar
  73. Wilson DF (1967) Effect of temperature on the spectral properties of some ferrocytochromes. Arch Biochem Biophys 121:757–768CrossRefPubMedGoogle Scholar
  74. Zhang D, Su D, Bérczi A, Vargas A, Asard H (2006) An ascorbate-reducible cytochrome b561 is localized in macrophage lysosomes. Biochim Biophys Acta 1760:1903–1913PubMedGoogle Scholar
  75. Zopellaro G, Bren KL, Ensign AA, Herbitz E, Kaur R, Hersleth HP, Ryde U, Hederstedt L, Andersson KK (2009) Studies of ferric heme proteins with highly anisotropic/highly axial low spin (S = 1/2) electron paramagnetic resonance signals with bis-histidine and histidine-methionine axial iron coordination. Biopolymers. doi:  10.1002/bip.21267

Copyright information

© European Biophysical Societies' Association 2009

Authors and Affiliations

  • Alajos Bérczi
    • 1
  • Filip Desmet
    • 2
  • Sabine Van Doorslaer
    • 2
  • Han Asard
    • 3
  1. 1.Institute of Biophysics, Biological Research CenterHungarian Academy of SciencesSzegedHungary
  2. 2.Department of PhysicsUniversity of AntwerpWilrijkBelgium
  3. 3.Department of BiologyUniversity of AntwerpAntwerpenBelgium

Personalised recommendations