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The reduction rates of DEPC-modified mutant Thermus thermophilus Rieske proteins differ when there is a negative charge proximal to the cluster

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Abstract

Rieske and Rieske-type proteins are electron transport proteins involved in key biological processes such as respiration, photosynthesis, and detoxification. They have a [2Fe–2S] cluster ligated by two cysteines and two histidines. A series of mutations, L135E, L135R, L135A, and Y158F, of the Rieske protein from Thermus thermophilus has been produced which probe the effects of the neighboring residues, in the second sphere, on the dynamics of cluster reduction and the reactivity of the ligating histidines. These properties were probed using titrations and modifications with diethyl pyrocarbonate (DEPC) at various pH values monitored using UV–Visible and circular dichroism spectrophotometry. These results, along with results from EPR studies, provide information on ligating histidine modification and rate of reduction of each of the mutant proteins. L135R, L135A, and Y158F react with DEPC similarly to wild type, resulting in modified protein with a reduced [2Fe–2S] cluster in <90 min, whereas L135E requires >15 h under the same conditions. Thus, the negative charge slows down the rate of reduction and provides an explanation as to why negatively charged residues are rarely, if ever, found in the equivalent position of other Rieske and Rieske-type proteins.

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Notes

  1. The pK a values for truncTtRp were previously determined [5], however, the experiment was repeated here with a higher concentration of buffer to overcome any effects the protein storage buffer. Only small differences in pK a are observed. See Materials and Methods for more details.

  2. It is notable that for truncTtRp, the maximum absorbance difference was at 250 nm [12], whereas for each of the mutants reported here, the absorbance difference maximum was at or near 240 nm.

    .

  3. It is noteworthy that the Y158F signal is smaller than the other proteins. We suspect that the cluster incorporation for this protein was lower than the others and results in a smaller EPR signal.

Abbreviations

bc 1 :

The cytochrome bc 1 complex, complex III of the electron transport chain

CAPS:

3-(Cyclohexylamino)-1-propanesulfonic acid

CD:

Circular dichroism

CW-EPR:

Continuous-wave electron paramagnetic resonance

DEPC:

Diethyl pyrocarbonate

DMSO:

Dimethyl sulfoxide

HEPES:

4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid

IPTG:

Isopropyl β-d-1-thiogalactopyranoside

LMCT:

Ligand-to-metal charge transfer

MES:

2-(N-Morpholino)ethanesulfonic acid hydrate

MOPS:

3-(N-Morpholino)propanesulfonic acid

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

TAPS:

N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid

Tris:

2-Amino-2-(hydroxymethyl)-1,3-propanediol

truncTtRp:

The truncated version of the Rieske protein from Thermus thermophilus

TtRp:

The Thermus thermophilus Rieske protein that has the additional 17 amino acids

References

  1. Link TA (1999) The structures of Rieske and Rieske-type proteins. Adv Inorg Chem 47:83–157

    Article  CAS  Google Scholar 

  2. Hunsicker-Wang LM, Heine A, Chen Y, Luna EP, Todaro T, Zhang Y, Williams PA, McRee DE, Hirst J, Stout CD, Fee JA (2003) High resolution structure of the soluble, respiratory-type Rieske protein from Thermus thermophilus: analysis and comparison. Biochemistry 42:7303–7317

    Article  PubMed  CAS  Google Scholar 

  3. Link TA (2001) Fe-S Rieske center. In: Messerschmidt A, Huber R, Weighardt K, Poulos T (eds) Handbook of metalloproteins, vol 1. Wiley, New York, pp 518–531

    Google Scholar 

  4. Hsueh K, Westler WM, Markley JL (2010) NMR investigations of the Rieske protein from Thermus thermophilus support a coupled proton and electron transfer mechanism. J Am Chem Soc 132:7908–7918

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  5. Konkle ME, Muellner SK, Schwander AL, Dicus MM, Pokhrel R, Britt RD, Taylor AB, Hunsicker-Wang LM (2009) Effect of pH on the Rieske protein from Thermus thermophilus: a spectroscopic and structural analysis. Biochemistry 48:9848–9857

    Article  PubMed  CAS  Google Scholar 

  6. Iwata S, Lee JW, Okada K, Lee JK, Iwata M, Rasmussen B, Link TA, Ramaswamy S, Jap BK (1998) Complete structure of the 11-subunit bovine mitochondrial cytochrome bc1 complex. Science 281:64–71

    Article  PubMed  CAS  Google Scholar 

  7. Esser L, Quinn B, Li YF, Zhang M, Elberry M, Yu L, Yu CA, Xia D (2004) Crystallographic studies of quinol oxidation site inhibitors: a modified classification of inhibitors for the cytochrome bc(1) complex. J Mol Biol 341:281–302

    Article  PubMed  CAS  Google Scholar 

  8. Gurung B, Yu L, Yu CA (2008) Stigmatellin induces reduction of iron–sulfur protein in the oxidized cytochrome bc1 complex. J Biol Chem 283:28087–28094

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  9. von Jagow G, Ohnishi T (1985) The chromone inhibitor stigmatellin-binding to the ubiquinol oxidation center at the C-side of the mitochondrial membrane. FEBS Lett 185:311–315

    Article  Google Scholar 

  10. Zu Y, Fee JA, Hirst J (2001) Complete thermodynamic characterization of reduction and protonation of the bc 1-type Rieske [2Fe–2S] center of Thermus thermophilus. J Am Chem Soc 123:9906–9907

    Article  PubMed  CAS  Google Scholar 

  11. Zu Y, Couture MM, Kolling DRJ, Crofts AR, Eltis LD, Fee JA, Hirst J (2003) Reduction potentials of Rieske clusters: importance of the coupling between oxidation state and histidine protonation state. Biochemistry 42:12400–12408

    Article  PubMed  CAS  Google Scholar 

  12. Konkle ME, Elsenheimer KN, Hakala K, Robicheaux JC, Weintraub ST, Hunsicker-Wang LM (2010) Chemical modification of the Rieske protein from Thermus thermophilus using diethyl pyrocarbonate modifies ligating histidine 154 and reduces the [2Fe–2S] cluster. Biochemistry 49:7272–7281

    Article  PubMed  CAS  Google Scholar 

  13. Lin I, Chen Y, Fee JA, Song J, Westler WM, Markley JL (2006) Rieske protein from Thermus thermophilus: 15N NMR titration study demonstrates the role of iron-ligated histidines in the pH dependence of the reduction potential. J Am Chem Soc 128:10672–10673

    Article  PubMed  CAS  Google Scholar 

  14. Iwaki M, Yakovlev G, Hirst J, Osyczka A, Dutton PL, Marshall D, Rich PR (2005) Direct observation of redox-linked histidine protonation changes in the iron–sulfur protein of the cytochrome bc 1 complex by ATR-FTIR spectroscopy. Biochemistry 44:4230–4237

    Article  PubMed  CAS  Google Scholar 

  15. Miles EW (1977) Modification of histidyl residues in proteins by diethylpyrocarbonate. Methods Enzymol 47:431–442

    Article  PubMed  CAS  Google Scholar 

  16. Lundblad RL (2005) Chemical reagents for protein modification, 3rd edn. CRC Press, Boca Raton

    Google Scholar 

  17. Yagi T, Vik SB, Hatefi Y (1982) Reversible inhibition of the mitochondrial ubiquinol-cytochrome c oxidoreductase complex (complex III) by ethoxyformic anhydride. Biochemistry 21:4777–4782

    Article  PubMed  CAS  Google Scholar 

  18. Ohnishi T, Meinhardt SW, von Jagow G, Yagi T, Hatefi Y (1994) Effect of ethoxyformic anhydride on the Rieske iron–sulfur protein of bovine heart ubiquinol: cytochrome c oxidoreductase. FEBS Lett 353:103–107

    Article  PubMed  CAS  Google Scholar 

  19. Lorusso M, Gatti D, Boffoli D, Bellomo E, Papa S (1983) Redox-linked proton translocation in the b–c1 complex from beef-heart mitochondria reconstituted into phospholipid vesicles. Studies with chemical modifiers of amino acid residues. Eur J Biochem 137:413–420

    Article  PubMed  CAS  Google Scholar 

  20. Lorusso M, Gatti D, Marzo M, Boffoli D, Cocco T, Papa S (1987) Chemical modification studies of beef-heart mitochondrial b–c1 complex. Effect of modification by ethoxyformic anhydride. Eur J Biochem 162:231–238

    Article  PubMed  CAS  Google Scholar 

  21. Zu Y, Fee JA, Hirst J (2002) Breaking and re-forming the disulfide bond at the high-potential, respiratory-type Rieske [2Fe–2S] center of Thermus thermophilus: characterization of the sulfhydryl state by protein-film voltammetry. Biochemistry 41:14054–14065

    Article  PubMed  CAS  Google Scholar 

  22. Pace CN, Vajdos F, Fee L, Grimsley G, Gray T (1995) How to measure and predict the molar absorption coefficient of a protein. Protein Sci 4:2411–2423

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  23. Edelhoch H (1967) Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry 6:1948–1954

    Article  PubMed  CAS  Google Scholar 

  24. Leggate EJ, Hirst J (2005) Roles of the disulfide bond and adjacent residues in determining the reduction potentials and stabilities of respiratory-type Rieske clusters. Biochemistry 44:7048–7058

    Article  PubMed  CAS  Google Scholar 

  25. Klingen AR, Ullmann GM (2004) Negatively charged residues and hydrogen bonds tune the ligand histidine pKa values of Rieske iron–sulfur proteins. Biochemistry 43:12383–12389

    Article  PubMed  CAS  Google Scholar 

  26. Kolling DJ, Brunzelle JS, Lhee S, Crofts AR, Nair SK (2007) Atomic resolution structures of Rieske iron–sulfur protein: role of hydrogen bonds in tuning the redox potential of iron–sulfur clusters. Structure 15:29–38

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  27. Kuila D, Fee JA (1986) Evidence for a redox-linked ionizable group associated with the [2Fe–2S] cluster of the Thermus Rieske protein. J Biol Chem 261:2768–2771

    PubMed  CAS  Google Scholar 

  28. Link TA, Hatzfeld OM, Unalkat P, Shergill JK, Cammack R, Mason JR (1996) Comparison of the “Rieske” [2Fe–2S] center in the bc 1 complex and in bacterial dioxygenases by circular dichroism spectroscopy and cyclic voltammetry. Biochemistry 35:7546–7552

    Article  PubMed  CAS  Google Scholar 

  29. Bertrand P, Guigliarelli B, Gayda J, Peter B, Gibson JF (1985) A ligand-field model to describe a new class of 2Fe–2S clusters in proteins and their synthetic analogues. Biochimica et Biophysica Acta (BBA) Protein Struct Mol Enzymol 831:261–266

  30. Orio M, Mouesca JM (2008) Variation of average g values and effective exchange coupling constants among [2Fe–2S] clusters: a density functional theory study of the impact of localization (trapping forces) versus delocalization (double-exchange) as competing factors. Inorg Chem 47:5394–5416

    Article  PubMed  CAS  Google Scholar 

  31. Denke E, Merbitz-Zahradnik T, Hatzfeld OM, Snyder CH, Link TA, Trumpower BL (1998) Alteration of the midpoint potential and catalytic activity of the Rieske iron–sulfur protein by changes of amino acids forming hydrogen bonds to the iron–sulfur cluster. J Biol Chem 273:9085–9093

    Article  PubMed  CAS  Google Scholar 

  32. Grace ME, Loosemore MJ, Semmel ML, Pratt RF (1980) Kinetics and mechanism of Bamberger cleavage of imidazole and of histidine derivatives by diethyl pyrocarbonate in aqueous solution. J Am Chem Soc 102:6784–6789

    Article  CAS  Google Scholar 

  33. Iwasaki T, Fukazawa R, Miyajima-Nakano Y, Baldansuren A, Matsushita S, Lin MT, Gennis RB, Hasegawa K, Kumasaka T, Dikanov SA (2012) Dissection of hydrogen bond interaction network around an iron–sulfur cluster by site-specific isotope labeling of hyperthermophilic archaeal Rieske-type ferredoxin. J Am Chem Soc 134:19731–19738

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  34. Holm L, Rosenström P (2010) Dali server: conservation mapping in 3D. Nucleic Acids Res 38:W545–W549

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  35. Kauppi B, Lee K, Carredano E, Parales RE, Gibson DT, Eklund H, Ramaswamy S (1998) Structure of an aromatic-ring-hydroxylating dioxygenase-naphthalene 1,2-dioxygenase. Structure 6:571–586

    Article  PubMed  CAS  Google Scholar 

  36. Dong X, Fushinobu S, Fukuda E, Terada T, Nakamura S, Shimizu K, Nojiri H, Omori T, Shoun H, Wakagi T (2005) Crystal structure of the terminal oxygenase component of cumene dioxygenase from pseudomonas fluorescens IP01. J Bacteriol 187:2483–2490

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  37. Gouet P, Courcelle E, Stuart DI, Metoz F (1999) ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics 15:305–308

    Article  PubMed  CAS  Google Scholar 

  38. Carrell CJ, Zhang H, Cramer WA, Smith JL (1997) Biological identity and diversity in photosynthesis and respiration: structure of the lumen-side domain of the chloroplast Rieske protein. Structure 5:1613–1625

    Article  PubMed  CAS  Google Scholar 

  39. Veit S, Takeda K, Tsunoyama Y, Rexroth D, Rogner M, Miki K (2012) Structure of a thermophilic cyanobacterial b6f-type Rieske protein. Acta Crystallogr D Biol Crystallogr 68:1400–1408

    Article  PubMed  CAS  Google Scholar 

  40. Iwata S, Saynovits M, Link TA, Michel H (1996) Structure of a water soluble fragment of the ‘Rieske’ iron–sulfur protein of the bovine heart mitochondrial cytochrome bc1 complex determined by MAD phasing at 1.5 A resolution. Structure 4:567–579

    Article  PubMed  CAS  Google Scholar 

  41. Bonisch H, Schmidt CL, Schafer G, Ladenstein R (2002) The structure of the soluble domain of an archaeal Rieske iron–sulfur protein at 1.1 Å resolution. J Mol Biol 319:791–805

    Article  PubMed  CAS  Google Scholar 

  42. Friemann R, Lee K, Brown EN, Gibson DT, Eklund H, Ramaswamy S (2009) Structures of the multicomponent Rieske non-heme iron toluene 2,3-dioxygenase enzyme system. Acta Crystallogr D Biol Crystallogr 65:24–33

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  43. Ashikawa Y, Fujimoto Z, Usami Y, Inoue K, Noguchi H, Yamane H, Nojiri H (2012) Structural insight into the substrate- and dioxygen-binding manner in the catalytic cycle of Rieske nonheme iron oxygenase system, carbazole 1,9a-dioxygenase. BMC Struct Biol 12:15. doi:10.1186/1472-6807-12-15

  44. Colbert CL, Couture MM, Eltis LD, Bolin JT (2000) A cluster exposed: structure of the Rieske ferredoxin from biphenyl dioxygenase and the redox properties of Rieske Fe–S proteins. Structure 8:1267–1278

    Article  PubMed  CAS  Google Scholar 

  45. Brown EN, Friemann R, Karlsson A, Parales JV, Couture MM, Eltis LD, Ramaswamy S (2008) Determining Rieske cluster reduction potentials. J Biol Inorg Chem 13:1301–1313

    Article  PubMed  CAS  Google Scholar 

  46. Ellis PJ, Conrads T, Hille R, Kuhn P (2001) Crystal structure of the 100 kDa arsenite oxidase from Alcaligenes faecalis in two crystal forms at 1.64 Å and 2.03 Å. Structure 9:125–132

    Article  PubMed  CAS  Google Scholar 

  47. Moe LA, Bingman CA, Wesenberg GE, Phillips GN Jr, Fox BG (2006) Structure of T4moC, the Rieske-type ferredoxin component of toluene 4-monooxygenase. Acta Crystallogr D Biol Crystallogr 62:476–482

    Article  PubMed  Google Scholar 

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Acknowledgments

This work was supported by funds from the National Science Foundation (CHE-1058273) and the Welch Foundation (W-0031). Funding for EMW was provided in part by a grant from the Howard Hughes Medical Institute. We would like to acknowledge the work of Ravi Pokhrel who made the L135A mutant and Sarah Muellner who made the Y158F mutant. We would also like to acknowledge Abhishek Chhetri who discovered the need to change the buffer conditions for the pH-dependent UV–Visible titrations. We would also like to thank Drs. Bert Chandler and Nancy Mills for helpful discussions.

Author information

Author notes

  1. Nicholas E. Karagas

    Present address: University of Texas at Houston Medical School, 6431 Fannin Street, Houston, TX, 77225, USA

  2. Anika L. Dzierlenga

    Present address: Department of Pharmacology and Toxicology, University of Arizona, 1295 N. Martin, Tucson, AZ, 85721, USA

  3. Mary E. Konkle

    Present address: Department of Chemistry, Eastern Illinois University, 600 W. Lincoln Ave., Charleston, IL, 61920, USA

  4. Emily M. Whitney

    Present address: University of Georgia College of Pharmacy, 250 W. Green Street, Athens, GA, 30602, USA

Authors and Affiliations

  1. Department of Chemistry, Trinity University, One Trinity Place, San Antonio, TX, 78212, USA

    Nicholas E. Karagas, Christie N. Jones, Deborah J. Osborn, Anika L. Dzierlenga, Mary E. Konkle, Emily M. Whitney & Laura M. Hunsicker-Wang

  2. Department of Chemistry, University of California at Davis, One Shields Avenue, Davis, CA, 95616, USA

    Paul Oyala & R. David Britt

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Correspondence to Laura M. Hunsicker-Wang.

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Karagas, N.E., Jones, C.N., Osborn, D.J. et al. The reduction rates of DEPC-modified mutant Thermus thermophilus Rieske proteins differ when there is a negative charge proximal to the cluster. J Biol Inorg Chem 19, 1121–1135 (2014). https://doi.org/10.1007/s00775-014-1167-9

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