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The roles of C-terminal residues on the thermal stability and local heme environment of cytochrome c’ from the thermophilic purple sulfur bacterium Thermochromatium tepidum

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Abstract

A soluble cytochrome (Cyt) c’ from thermophilic purple sulfur photosynthetic bacterium Thermochromatium (Tch.) tepidum exhibits marked thermal tolerance compared with that from the closely related mesophilic counterpart Allochromatium vinosum. Here, we focused on the difference in the C-terminal region of the two Cyts c’ and examined the effects of D131 and R129 mutations on the thermal stability and local heme environment of Cyt c’ by differential scanning calorimetry (DSC) and resonance Raman (RR) spectroscopy. In the oxidized forms, D131K and D131G mutants exhibited denaturing temperatures significantly lower than that of the recombinant control Cyt c’. In contrast, R129K and R129A mutants denatured at nearly identical temperatures with the control Cyt c’, indicating that the C-terminal D131 is an important residue maintaining the enhanced thermal stability of Tch. tepidum Cyt c’. The control Cyt c’ and all of the mutants increased their thermal stability upon the reduction. Interestingly, D131K exhibited narrow DSC curves and unusual thermodynamic parameters in both redox states. The RR spectra of the control Cyt c’ exhibited characteristic bands at 1,635 and 1,625 cm−1, ascribed to intermediate spin (IS) and high spin (HS) states, respectively. The IS/HS distribution was differently affected by the D131 and R129 mutations and pH changes. Furthermore, R129 mutants suggested the lowering of their redox potentials. These results strongly indicate that the D131 and R129 residues play significant roles in maintaining the thermal stability and modulating the local heme environment of Tch. tepidum Cyt c’.

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Abbreviations

Cyt:

Cytochrome

Tch. :

Thermochromatium

Alc. :

Allochromatium

LH:

Light-harvesting

RC:

Reaction center

DSC:

Differential scanning calorimetry

RR:

Resonance Raman

References

  • Ambler RP, Bartsch RG, Daniel M, Kamen MD, McLellan L, Meyer TE, Van Beeumen J (1981) Amino acid sequences of bacterial cytochromes c’ and c-556. Proc Natl Acad Sci USA 78(11):6854–6857

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Benini S, Rypniewski WR, Wilson KS, Ciurli S (2008) High resolution crystal structure of Rubrivivax gelatinosus cytochrome c’. J Inorg Biochem 102(5–6):1322–1328

    Article  CAS  PubMed  Google Scholar 

  • Castillo MCG, Lou B-S, Ondrias MR, Robertson DE, Knaff DB (1994) Characterization of flavocytochrome c 552 from the thermophilic photosynthetic bacterium Chromatium tepidum. Arch Biochem Biophys 315:262–266

    Article  Google Scholar 

  • Cohen DS, Pielak GJ (1995) Entropic stabilization of cytochrome c upon reduction. J Am Chem Soc 117(6):1675–1677

    Article  CAS  Google Scholar 

  • Cusanovich MA (1971) Molecular weights of some cytochromes Cc’. Biochim Biophys Acta 236(1):238–241

    Article  CAS  PubMed  Google Scholar 

  • Cusanovich MA, Gibson QH (1973) Anomalous ligand binding by a class of high-spin c-type cytochromes. J Biol Chem 248(3):822–834

    CAS  PubMed  Google Scholar 

  • Desbois A (1994) Resonance Raman-spectroscopy of C-type cytochromes. Biochimie 76(7):693–707

    Article  CAS  PubMed  Google Scholar 

  • Desbois A, Lutz M (1981) Low-frequency vibrations of ferroprotoporphyrin-substituted imidazole complexes—a Resonance Raman-study. Biochim Biophys Acta 671(2):168–176

    Article  CAS  Google Scholar 

  • Doyle ML, Gill SJ, Cusanovich MA (1986) Ligand-controlled dissociation of Chromatium vinosum cytochrome-c’. Biochemistry 25(9):2509–2516

    Article  CAS  PubMed  Google Scholar 

  • Ehrenberg A, Kamen MD (1965) Magnetic and optical properties of some bacterial haem proteins. Biochim Biophys Acta 102(2):333–340

    Article  CAS  PubMed  Google Scholar 

  • Emptage MH, Zimmermann R, Que L Jr, Munck E, Hamilton WD, Orme-Johnson WH (1977) Mossbauer studies of cytochrome c’ from Rhodospirillum rubrum. Biochim Biophys Acta 495(1):12–23

    Article  CAS  PubMed  Google Scholar 

  • Finzel BC, Weber PC, Hardman KD, Salemme FR (1985) Structure of ferricytochrome c’ from Rhodospirillum-Molischianum at 1.67-Å resolution. J Mol Biol 186(3):627–643

    Article  CAS  PubMed  Google Scholar 

  • Fujii S, Masanari M, Inoue H, Yamanaka M, Wakai S, Nishihara H, Sambongi Y (2013) High thermal stability and unique trimer formation of cytochrome c’ from thermophilic Hydrogenophilus thermoluteolus. Biosci Biotech Bioch 77(8):1677–1681

    Article  CAS  Google Scholar 

  • Gibson QH, Kamen MD (1966) Kinetic analysis of reaction of cytochrome Cc’ with carbon monoxide. J Biol Chem 241(9):1969–1976

    CAS  PubMed  Google Scholar 

  • Higuchi M, Hirano Y, Kimura Y, Oh-oka H, Miki K, Wang ZY (2009) Overexpression, characterization, and crystallization of the functional domain of cytochrome c(z) from Chlorobium tepidum. Photosynth Res 102(1):77–84

    Article  CAS  PubMed  Google Scholar 

  • Hirano Y, Kimura Y, Suzuki H, Miki K, Wang ZY (2012) Structure analysis and comparative characterization of the cytochrome c’ and flavocytochrome c from thermophilic purple photosynthetic bacterium Thermochromatium tepidum. Biochemistry 51(33):6556–6567

    Article  CAS  PubMed  Google Scholar 

  • Hough MA, Antonyuk SV, Barbieri S, Rustage N, McKay AL, Servid AE, Eady RR, Andrew CR, Hasnain SS (2011) Distal-to-proximal NO conversion in hemoproteins: the role of the proximal pocket. J Mol Biol 405(2):395–409

    Article  CAS  PubMed  Google Scholar 

  • Huston WM, Andrew CR, Servid AE, McKay AL, Leech AP, Butler CS, Moir JWB (2006) Heterologous overexpression and purification of cytochrome c’ from Rhodobacter capsulatus and a mutant (K42E) in dimerization region. Mutation does not alter oligomerization but impacts the heme iron spin state and nitric oxide binding properties. Biochemistry 45:4388–4395

    Article  CAS  PubMed  Google Scholar 

  • Kamen MD, Kakuno T, Bartsch RG, Hannon S (1973) Spin-state correlations in near-infrared spectroscopy of cytochrome c’. Proc Natl Acad Sci USA 70(6):1851–1854

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Kassner RJ, Kykta MG, Cusanovich MA (1985) Binding of cyanide to cytochrome c’ from Chromatium vinosum. Biochim Biophys Acta 831:155–158

    Article  CAS  PubMed  Google Scholar 

  • Kekilli D, Dworkowski FSN, Pompidor G, Fuchs MR, Andrew CR, Antonyuk S, Strange RW, Eady RR, Hasnain SS, Hough MA (2014) Fingerprinting redox and ligand states in haemprotein crystal structures using resonance Raman spectroscopy. Acta Crystallogr D 70:1289–1296

    Article  CAS  PubMed  Google Scholar 

  • Kimura Y, Hirano Y, Yu LJ, Suzuki H, Kobayashi M, Wang ZY (2008) Calcium ions are involved in the unusual red shift of the light-harvesting 1 Q y transition of the core complex in thermophilic purple sulfur bacterium Thermochromatium tepidum. J Biol Chem 283(20):13867–13873

    Article  CAS  PubMed  Google Scholar 

  • Kimura Y, Yu LJ, Hirano Y, Suzuki H, Wang ZY (2009) Calcium ions are required for the enhanced thermal stability of the light-harvesting-reaction center core complex from thermophilic purple sulfur bacterium Thermochromatium tepidum. J Biol Chem 284(1):93–99

    Article  CAS  PubMed  Google Scholar 

  • Kitagawa T, Ozaki Y, Kyogoku Y, Horio T (1977) Resonance Raman study of pH-dependent and detergent-induced structural alterations in heme moiety of Rhodospirillum rubrum cytochrome-c’. Biochim Biophys Acta 495(1):1–11

    Article  CAS  PubMed  Google Scholar 

  • Kobayashi M, Fujioka Y, Mori T, Terashima M, Suzuki H, Shimada Y, Saito T, Wang ZY, Nozawa T (2005a) Reconstitution of photosynthetic reaction centers and core antenna-reaction center complexes in liposomes and their thermal stability. Biosci Biotech Bioch 69(6):1130–1136

    Article  CAS  Google Scholar 

  • Kobayashi M, Saito T, Takahashi K, Wang Z-Y, Nozawa T (2005b) Electronic properties and thermal stability of soluble redox proteins from a thermophilic purple sulfur photosynthetic bacterium, Thermochromatium tepidum. Bull Chem Soc Jpn 78:2164–2170

    Article  CAS  Google Scholar 

  • Lamar GN, Jackson JT, Bartsch RG (1981) Analysis of field-dependent relaxation data and hyperfine shifts of cytochrome-c’ from Rhodospirillum rubrum in terms of the high-spin iron ligation states. J Am Chem Soc 103(15):4405–4410

    Article  CAS  Google Scholar 

  • Lawson DM, Stevenson CEM, Andrew CR, Eady RR (2000) Unprecedented proximal binding of nitric oxide to heme: implications for guanylate cyclase. EMBO J 19(21):5661–5671

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Liu LJ, Nogi T, Kobayashi M, Nozawa T, Miki K (2002) Ultrahigh-resolution structure of high-potential iron-sulfur protein from Thermochromatium tepidum. Acta Crystallogr D 58:1085–1091

    Article  PubMed  Google Scholar 

  • Madigan MT (1984) A novel photosynthetic bacterium isolated from a Yellowstone hot spring. Science 225:313–315

    Article  CAS  PubMed  Google Scholar 

  • Maltempo MM (1974) Magnetic state of an unusual bacterial heme protein. J Chem Phys 61(7):2540–2547

    Article  CAS  Google Scholar 

  • Maltempo MM, Moss TH (1976) Spin 3/2 state and quantum spin mixtures in heme proteins. Q Rev Biophys 9(2):181–215

    Article  CAS  PubMed  Google Scholar 

  • Meyer TE, Kamen MD (1982) New perspectives on c-type cytochromes. Adv Protein Chem 35:105–212

    Article  CAS  PubMed  Google Scholar 

  • Meyer TE, Cheddar G, Bartsch RG, Getzoff ED, Cusanovich MA, Tollin G (1986) Kinetics of electron-transfer between cytochromes-c’ and the semiquinones of free flavin and clostridial flavodoxin. Biochemistry 25(6):1383–1390

    Article  CAS  PubMed  Google Scholar 

  • Moore GR, Pettigrew GW (1990) Cytochromes c : evolutionary, structural, and physicochemical aspects. Springer, Berlin

    Google Scholar 

  • Moulis JM, Scherrer N, Gagnon J, Forest E, Petillot Y, Garcia D (1993) Primary structure of Chromatium tepidum high-potential iron-sulfur protein in relation to thermal-denaturation. Arch Biochem Biophys 305(1):186–192

    Article  CAS  PubMed  Google Scholar 

  • Nagai K, Kitagawa T, Morimoto H (1980) Quaternary structures and low-frequency molecular vibrations of hemes of deoxy and oxyhemoglobin studied by resonance Raman-scattering. J Mol Biol 136(3):271–289

    Article  CAS  PubMed  Google Scholar 

  • Niwa S, Yu LJ, Takeda K, Hirano Y, Kawakami T, Wang-Otomo ZY, Miki K (2014) Structure of the LH1-RC complex from Thermochromatium tepidum at 3.0 angstrom. Nature 508(7495):228–232

    Article  CAS  PubMed  Google Scholar 

  • Othman S, Richaud P, Vermeglio A, Desbois A (1996) Evidence for a proximal histidine interaction in the structure of cytochromes c’ in solution: a resonance Raman study. Biochemistry 35(28):9224–9234

    Article  CAS  PubMed  Google Scholar 

  • Pixton DA, Petersen CA, Franke A, van Eldik R, Garton EM, Andrew CR (2009) Activation parameters for heme-NO binding in Alcaligenes xylosoxidans cytochrome c ‘: the putative dinitrosyl intermediate forms via a dissociative mechanism. J Am Chem Soc 131(13):4846–4853

    Article  CAS  PubMed  Google Scholar 

  • Ramirez LM, Axelrod HL, Herron SR, Rupp B, Allen JP, Kantardjieff KA (2003) High resolution crystal structure of ferricytochrome c’ from Rhodobacter sphaeroides. J Chem Crystallogr 33(5–6):413–424

    Article  CAS  Google Scholar 

  • Rawlings J, Stephens PJ, Nafie LA, Kamen MD (1977) Near-infrared magnetic circular-dichroism of cytochrome c’. Biochemistry 16(8):1725–1729

    Article  CAS  PubMed  Google Scholar 

  • Ren Z, Meyer T, Mcree DE (1993) Atomic-structure of a cytochrome-c’ with an unusual ligand-controlled dimer dissociation at 1.8 angstrom resolution. J Mol Biol 234(2):433–445

    Article  CAS  PubMed  Google Scholar 

  • Rubinow SC, Kassner RJ (1984) Cytochromes-c’ in their reaction with ethyl isocyanide. Biochemistry 23(12):2590–2595

    Article  CAS  PubMed  Google Scholar 

  • Sekine F, Horiguchi K, Kashino Y, Shimizu Y, Yu LJ, Kobayashi M, Wang ZY (2012) Gene sequencing and characterization of the light-harvesting complex 2 from thermophilic purple sulfur bacterium Thermochromatium tepidum. Photosynth Res 111(1–2):9–18

    Article  CAS  PubMed  Google Scholar 

  • Shibata N, Iba S, Misaki S, Meyer TE, Bartsch RG, Cusanovich MA, Morimoto Y, Higuchi Y, Yasuoka N (1998) Basis for monomer stabilization in Rhodopseudomonas palustris cytochrome c’ derived from the crystal structure. J Mol Biol 284(3):751–760

    Article  CAS  PubMed  Google Scholar 

  • Sot B, Banuelos S, Valpuesta JM, Muga A (2003) GroEL stability and function—contribution of the ionic interactions at the inter-ring contact sites. J Biol Chem 278(34):32083–32090

    Article  CAS  PubMed  Google Scholar 

  • Spiro TG, Strekas TC (1974) Resonance Raman-spectra of heme proteins—effects of oxidation and spin state. J Am Chem Soc 96(2):338–345

    Article  CAS  PubMed  Google Scholar 

  • Spiro TG, Czernuszewicz RS, Li XY (1990) Metalloporphyrin structure and dynamics from resonance Raman-spectroscopy. Coordin Chem Rev 100:541–571

    Article  CAS  Google Scholar 

  • Strekas TC, Spiro TG (1974) Resonance-Raman evidence for anomalous heme structures in cytochrome c’ from Rhodopseudomonas palustris. Biochim Biophys Acta 351(2):237–245

    Article  CAS  PubMed  Google Scholar 

  • Sturtevant JM (1987) Biochemical applications of differential scanning calorimetry. Annu Rev Phys Chem 38:463–488

    Article  CAS  Google Scholar 

  • Tahirov TH, Misaki S, Meyer TE, Cusanovich MA, Higuchi Y, Yasuoka N (1996) High-resolution crystal structures of two polymorphs of cytochrome c’ from the purple phototrophic bacterium Rhodobacter capsulatus. J Mol Biol 259(3):467–479

    Article  CAS  PubMed  Google Scholar 

  • Taniguchi S, Kamen MD (1963) On anomalous interactions of ligands with Rhodospirillum haem protein. Biochim Biophys Acta 74(3):438–455

    Article  CAS  PubMed  Google Scholar 

  • Tateishi Y, Abe T, Tamogami J, Nakao Y, Kikukawa T, Kamo N, Unno M (2011) Spectroscopic evidence for the formation of an N intermediate during the photocycle of sensory rhodopsin II (Phoborhodopsin) from Natronobacterium pharaonis. Biochemistry 50(12):2135–2143

    Article  CAS  PubMed  Google Scholar 

  • Teraoka J, Kitagawa T (1980) Raman characterization of axial ligands for pentacoordinate and hexacoordinate ferric high-spin and intermediate-spin (Octaethylporphyrinato)iron(III) complexes—elucidation of unusual resonance Raman-spectra of cytochrome C’. J Phys Chem 84(15):1928–1935

    Article  CAS  Google Scholar 

  • Tezcan FA, Winkler JR, Gray HB (1998) Effects of ligation and folding on reduction potentials of heme proteins. J Am Chem Soc 120(51):13383–13388

    Article  CAS  Google Scholar 

  • Watson AJ, Hughes AV, Fyfe PK, Wakeham MC, Holden-Dye K, Heathcote P, Jones MR (2005) On the role of basic residues in adapting the reaction centre—LH1 complex for growth at elevated temperatures in purple bacteria. Photosynth Res 86(1–2):81–100

    Article  CAS  PubMed  Google Scholar 

  • Weber PC (1982) Correlations between structural and spectroscopic properties of the high-spin heme protein cytochrome-c’. Biochemistry 21(21):5116–5119

    Article  CAS  PubMed  Google Scholar 

  • Weiss R, Gold A, Terner J (2006) Cytochromes c’: biological models for the S = (3)/(2), (5)/(2) spin-state admixture? Chem Rev 106(6):2550–2579

    Article  CAS  PubMed  Google Scholar 

  • Yoshimura T, Fujii S, Kamada H, Yamaguchi K, Suzuki S, Shidara S, Takakuwa S (1996) Spectroscopic characterization of nitrosylheme in nitric oxide complexes of ferric and ferrous cytochrome c’ from photosynthetic bacteria. Biochim Biophys Acta 1292(1):39–46

    Article  Google Scholar 

  • Yu LJ, Unno M, Kimura Y, Yanagimoto K, Oh-oka H, Wang-Otomo ZY (2013) Structure analysis and characterization of the cytochrome c-554 from thermophilic green sulfur photosynthetic bacterium Chlorobaculum tepidum. Photosynth Res 118(3):249–258

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported by Grants-in-aid for Scientific Research (C) (24570158) (Y.K.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We appreciate the Instrument Center of the Institute for Molecular Science for assistance of DSC measurements.

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Authors and Affiliations

  1. Department of Agrobioscience, Graduate School of Agricultural Science, Kobe University, Nada, Kobe, 657-8501, Japan

    Yukihiro Kimura, Sachiko Kasuga, Yuko Sasaki & Takashi Ohno

  2. Department of Chemistry and Applied Chemistry, Graduate School of Science and Engineering, Saga University, Saga, 840-8502, Japan

    Masashi Unno & Shinsuke Osoegawa

  3. Faculty of Science, Ibaraki University, 2-1-1 Bunkyo, Mito, 310-8512, Japan

    Takashi Furusawa & Zheng-Yu Wang-Otomo

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  1. Yukihiro Kimura
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  2. Sachiko Kasuga
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Correspondence to Yukihiro Kimura or Zheng-Yu Wang-Otomo.

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Kimura, Y., Kasuga, S., Unno, M. et al. The roles of C-terminal residues on the thermal stability and local heme environment of cytochrome c’ from the thermophilic purple sulfur bacterium Thermochromatium tepidum . Photosynth Res 124, 19–29 (2015). https://doi.org/10.1007/s11120-014-0069-6

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