Skip to main content
SpringerLink
Log in

Electronic structure of the Mn-cofactor of modified bacterial reaction centers measured by electron paramagnetic resonance and electron spin echo envelope modulation spectroscopies

  • Regular Paper
  • Published:
Photosynthesis Research Aims and scope Submit manuscript

Abstract

The electronic structure of a Mn(II) ion bound to highly oxidizing reaction centers of Rhodobacter sphaeroides was studied in a mutant modified to possess a metal binding site at a location comparable to the Mn4Ca cluster of photosystem II. The Mn-binding site of the previously described mutant, M2, contains three carboxylates and one His at the binding site (Thielges et al., Biochemistry 44:389–7394, 2005). The redox-active Mn-cofactor was characterized using electron paramagnetic resonance (EPR) and electron spin echo envelope modulation (ESEEM) spectroscopies. In the light without bound metal, the Mn-binding mutants showed an EPR spectrum characteristic of the oxidized bacteriochlorophyll dimer and reduced quinone whose intensity was significantly reduced due to the diminished quantum yield of charge separation in the mutant compared to wild type. In the presence of the metal and in the dark, the EPR spectrum measured at the X-band frequency of 9.4 GHz showed a distinctive spin 5/2 Mn(II) signal consisting of 16 lines associated with both allowed and forbidden transitions. Upon illumination, the amplitude of the spectrum is decreased by over 80 % due to oxidation of the metal upon electron transfer to the oxidized bacteriochlorophyll dimer. The EPR spectrum of the Mn-cofactor was also measured at the Q-band frequency of 34 GHz and was better resolved as the signal was composed of the six allowed electronic transitions with only minor contributions from other transitions. A fit of the Q-band EPR spectrum shows that the Mn-cofactor is a high spin Mn(II) species (S = 5/2) that is six-coordinated with an isotropic g-value of 2.0006, a weak zero-field splitting and E/D ratio of approximately 1/3. The ESEEM experiments showed the presence of one 14N coordinating the Mn-cofactor. The nitrogen atom is assigned to a His by comparing our ESEEM results to those previously reported for Mn(II) ions bound to other proteins and on the basis of the X-ray structure of the M2 mutant that shows the presence of only one His, residue M193, that can coordinate the Mn-cofactor. Together, the data allow the electronic structure and coordination environment of the designed Mn-cofactor in the modified reaction centers to be characterized in detail and compared to those observed in other proteins with Mn-cofactors.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

Abbreviations

P:

Bacteriochlorophyll dimer

QA :

Primary quinone

QB :

Secondary quinone

EPR:

Electron paramagnetic resonance

ESEEM:

Electron spin echo envelope modulation

hfc:

Hyperfine coupling

References

  • Allen JP, Williams JC (1995) Relationship between the oxidation potential of the bacteriochlorophyll dimer and electron transfer in photosynthetic reaction centers. J Bioenerg Biomembr 27:275–283

    Article  CAS  PubMed  Google Scholar 

  • Allen JP, Williams JC (2011) The evolutionary pathway from anoxygenic to oxygenic photosynthesis examined by comparison of the properties of photosystem II and bacterial reaction centers. Photosynth Res 107:59–69

    Article  CAS  PubMed  Google Scholar 

  • Allen JP, Williams JC, Graige MS, Paddock ML, Labahn A, Feher G, Okamura MY (1998) Free energy dependence of the direct charge recombination from the primary and secondary quinones in reaction centers from Rhodobacter sphaeroides. Photosynth Res 55:227–233

    Article  CAS  Google Scholar 

  • Allen JP, Olson TL, Oyala P, Lee WJ, Tufts AA, Williams JC (2012) Light-driven oxygen production from superoxide by Mn-binding bacterial reaction centers. Proc Natl Acad Sci USA 109:2314–2318

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Angerhofer A, Moomaw EW, Garcia-Rubio I, Ozarowski A, Krzystek J, Weber RT, Richards NGJ (2007) Multifrequency EPR studies on the Mn(II) centers of oxalate decarboxylase. J Phys Chem B 111:5043–5046

    Article  CAS  PubMed  Google Scholar 

  • Artz K, Williams JC, Allen JP, Lendzian F, Rautter J, Lubitz W (1997) Relationship between the oxidation potential and electron spin density of the primary electron donor in reaction centers from Rhodobacter sphaeroides. Proc Natl Acad Sci USA 94:13582–13587

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Begley TP, Ealick SE (2004) Enzymatic reactions involving novel mechanisms of carbanion stabilization. Curr Opin Chem Biol 8:508–515

    Article  CAS  PubMed  Google Scholar 

  • Beharry Z, Palzkill T (2005) Functional analysis of active site residues of the fosfomycin resistance enzyme FosA from Pseudomonas aeruginosa. J Biol Chem 280:17786–17791

    Article  CAS  PubMed  Google Scholar 

  • Burghaus O, Plato M, Bumann D, Neumann B, Lubitz W, Möbius K (1991) 3 mm EPR investigation of the primary donor cation radical P865 + in single crystals of Rhodobacter sphaeroides R-26 reaction centers. Chem Phys Lett 185:381–386

    Article  CAS  Google Scholar 

  • Butler WF, Calvo R, Fredkin DR, Isaacson RA, Okamura MY, Feher G (1984) The electronic structure of Fe2+ in reaction centers from Rhodopseudomonas sphaeroides. Biophys J 45:947–973

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Buy C, Girault G, Zimmermann JL (1996) Metal binding sites of H+ -ATPase from chloroplast and Bacillus PS3 studied by EPR and Pulsed EPR spectroscopy of bound manganese(II). Biochemistry 35:9880–9891

    Article  CAS  PubMed  Google Scholar 

  • Campbell KA, Yikilmaz E, Grant CV, Gregor W, Miller AF, Britt RD (1999) Parallel polarization EPR characterization of the Mn(III) center of oxidized manganese superoxide dismutase. J Am Chem Soc 121:4714–4715

    Article  CAS  Google Scholar 

  • Coffino AR, Peisach J (1996) Simulation of Mn(II) EPR spectra using a full spin-Hamiltonian approach. J Mag Reson B 111:127–134

    Article  CAS  Google Scholar 

  • Daubric H, Berger R, Kliava J, Chastanet G, Nguyen O, Letard J-F (2002) Light-induced excited spin-state trapping of Fe2+ observed by electron paramagnetic resonance of Mn2+. Phys Rev B 66:054423-1–054423-8

    Article  Google Scholar 

  • Deligiannakis Y, Louloudi M, Hadjiliadis N (2000) Electron spin echo envelope modulation (ESEEM) spectroscopy as a tool to investigate the coordination environment of metal centers. Coord Chem Rev 204:1–112

    Article  CAS  Google Scholar 

  • Espe MP, Hosler JP, Ferguson-Miller S, Babcock GT, McCracken J (1995) A continuous wave and pulsed EPR characterization of the Mn2+ binding site in Rhodobacter sphaeroides cytochrome c oxidase. J Biochem 34:7593–7602

    Article  CAS  Google Scholar 

  • Flores M, Isaacson RA, Calvo R, Feher G, Lubitz W (2003) Probing hydrogen bonding to quinone anion radicals by 1H and 2H ENDOR spectroscopy at 35 GHz. Chem Phys 294:401–413

    Article  CAS  Google Scholar 

  • Flores M, Isaacson R, Abresch E, Calvo R, Lubitz W, Feher G (2007) Protein-cofactor interactions in bacterial reaction centers from Rhodobacter sphaeroides R-26: II. Geometry of the hydrogen bonds to the primary quinone Q −A by 1H and 2H ENDOR spectroscopy. Biophys J 92:671–682

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Halkides CJ, Farrar CT, Singel DJ (1998) The effects of cryoprotection on the structure and activity of p21 ras: implications for electron spin-echo envelope modulation spectroscopy. J Mag Reson 134:142–153

    Article  CAS  Google Scholar 

  • Hunsicker-Wang L, Vogt M, DeRose VJ (2009) EPR methods to study specific metal-ion binding sites in RNA. Methods Enzymol 468:335–367

    Article  CAS  PubMed  Google Scholar 

  • Hunter CN, Daldal F, Thurnauer MC, Beatty JT (eds) (2009) The purple phototrophic bacteria. Springer, Dordrecht

    Google Scholar 

  • Jackson TA, Xie J, Yikilmaz E, Miller A-F, Brunold TC (2002) Spectroscopic and computational studies on iron and manganese superoxide dismutases: nature of the chemical events associated with active-site pKs. J Am Chem Soc 124:10833–10845

    Article  CAS  PubMed  Google Scholar 

  • Kálmán L, LoBrutto R, Allen JP, Williams JC (1999) Modified reaction centres oxidize tyrosine in reactions that mirror photosystem II. Nature 402:696–699

    Article  Google Scholar 

  • Kálmán L, Narváez AJ, LoBrutto R, Williams JC, Allen JP (2004) Dependence of tyrosine oxidation in highly oxidizing bacterial reaction centers on pH and free-energy difference. Biochemistry 43:12905–12912

    Article  PubMed  Google Scholar 

  • Kálmán L, Williams JC, Allen JP (2005a) Mimicking the properties of the oxygen-evolving complex in purple bacterial reaction centers. In: Wydrzynski T, Satoh K (eds) Photosystem II: the light-driven water: plastoquinone oxidoreductase. Springer, Dordrecht, pp 715–727

    Google Scholar 

  • Kálmán L, Thielges MC, Williams JC, Allen JP (2005b) Proton release due to manganese binding and oxidation in modified bacterial reaction centers. Biochemistry 44:13266–13273

    Article  PubMed  Google Scholar 

  • Kálmán L, Flores M, Williams JC, Allen JP (2010) Electronic structure of Fe3+ at a metal-binding site introduced in modified bacterial reaction centers. Appl Magn Reson 37:27–37

    Article  Google Scholar 

  • Kálmán L, Williams JC, Allen JP (2011) Energetics for oxidation of a bound manganese cofactor in modified bacterial reaction centers. Biochemistry 50:3310–3320

    Article  PubMed  Google Scholar 

  • Lin X, Murchison HA, Nagarajan V, Parson WW, Allen JP, Williams JC (1994) Specific alteration of the oxidation potential of the electron donor in reaction centers from Rhodobacter sphaeroides. Proc Natl Acad Sci USA 91:10265–10269

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • LoBrutto R, Smithers GW, Reed GH, Orme-Johnson WH, Tan SL, Leigh JS (1986) Observation of manganese(II)-ligand superhyperfine couplings in complexes with proteins by electron spin-echo spectroscopy. Biochemistry 25:5654–5660

    Article  CAS  PubMed  Google Scholar 

  • McCracken J, Peisach J, Bhattacharyya L, Brewer F (1991) Electron spin echo envelope modulation studies of lectins: evidence for a conserved Mn2+-binding site. Biochemistry 30:4486–4491

    Article  CAS  PubMed  Google Scholar 

  • McElroy JD, Feher G, Mauzerall DC (1969) On the nature of the free radical formed during the primary process of bacterial photosynthesis. Biochim Biophys Acta 172:180–183

    Article  CAS  PubMed  Google Scholar 

  • Miller A-F (2008) Redox tuning over almost 1 V in a structurally conserved active site: lessons from Fe-containing superoxide dismutase. Acc Chem Res 41:501–510

    Article  CAS  PubMed  Google Scholar 

  • Moomaw EW, Angehofer A, Moussatche P, Ozarowski A, Garcia-Rubio I, Richards NGJ (2009) Metal dependence of oxalate decarboxylase activity. Biochemistry 48:6116–6125

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Muh F, Lendzian F, Roy M, Williams JC, Allen JP, Lubitz W (2002) Pigment-protein interactions in bacterial reaction centers and their influence on oxidation potential and spin density distribution of the primary donor. J Phys Chem B 106:3226–3236

    Article  Google Scholar 

  • Narváez AJ, LoBrutto R, Allen JP, Williams JC (2004) Trapped tyrosyl radical populations in modified reaction centers from Rhodobacter sphaeroides. Biochemistry 43:14379–14384

    Article  PubMed  Google Scholar 

  • Okamura MY, Paddock ML, Graige MS, Feher G (2000) Proton and electron transfer in bacterial reaction centers. Biochim Biophys Acta 1458:148–163

    Article  CAS  PubMed  Google Scholar 

  • Perry JJP, Shin DS, Getzoff ED, Tainer JA (2010) The structural biochemistry of the superoxide dismutases. Biochim Biophys Acta 1804:245–262

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Reed GH, Markham GD (1984) EPR of Mn(II) complexes with enzymes and other proteins. Biol Mag Res 6:73–142

    Article  CAS  Google Scholar 

  • Rife CL, Pharris RE, Newcomer ME, Armstrong RN (2002) Crystal structure of a genomically encoded fosfomycin resistance protein (FosA) at 1.19 Å resolution by MAD phasing off the L-III edge of Tl+. J Am Chem Soc 124:11001–11003

    Article  CAS  PubMed  Google Scholar 

  • Sigel A, Sigel H (eds) (2000) Manganese and its role in biological processes. Marcel Dekker, New York

    Google Scholar 

  • Stich TA, Lahiri S, Yeagle G, Dicus M, Brynda M, Gunn A, Aznar C, DeRose VJ, Britt RD (2007) Multifrequency pulsed EPR studies of biologically relevant manganese(II) complexes. Appl Magn Reson 31:321–341

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Stoll S, Schweiger A (2006) EasySpin, a comprehensive software package for spectral simulation and analysis in EPR. J Magn Reson 178:42–55

    Article  CAS  PubMed  Google Scholar 

  • Svensson-Ek M, Abramson J, Larsson G, Tornroth S, Brzezinski P, Iwata S (2002) The X-ray crystal structures of wild-type and EQ(I-286) mutant cytochrome c oxidases from Rhodobacter sphaeroides. J Mol Biol 231:329–339

    Article  Google Scholar 

  • Tabares LC, Gatjens J, Un S (2010) Understanding the influence of the protein environment on the Mn(II) centers in superoxide dismutases using high-field electron paramagnetic resonance. Biochim Biophys Acta 1804:308–317

    Article  CAS  PubMed  Google Scholar 

  • Tan X, Bernardo M, Thomann H, Scholes CP (1995) 17O Hyperfine and quadrupole interactions for water ligands in frozen solutions of high spin Mn2+. J Chem Phys 102:2675–2690

    Article  CAS  Google Scholar 

  • Tang K, Williams JC, Allen JP, Kálmán L (2009) Effect of anions on the binding and oxidation of divalent manganese and iron in modified bacterial reaction centers. Biophysical J 96:3295–3304

    Article  CAS  Google Scholar 

  • Thielges M, Uyeda G, Camara-Artigas A, Kálmán L, Williams JC, Allen JP (2005) Design of a redox-linked active metal site: manganese bound to bacterial reaction centers at a site resembling that of photosystem II. Biochemistry 44:7389–7394

    Article  CAS  PubMed  Google Scholar 

  • Tipton PA, Peisach J (1991) Pulsed EPR analysis of tartrate dehydrogenase active-site complexes. Biochemistry 30:739–744

    Article  CAS  PubMed  Google Scholar 

  • Un S, Dorlet P, Voyard G, Tabares LC, Cortez N (2001) High-Field EPR Characterization of manganese reconstituted superoxide dismutase from Rhodobacter capsulatus. J Am Chem Soc 123:10123–10124

    Article  CAS  PubMed  Google Scholar 

  • Weil JA, Bolton JR (2007) Electron paramagnetic resonance: Elementary theory and practical applications. Wiley, Hoboken

    Google Scholar 

  • Williams JC, Taguchi AKW (1995) Genetic manipulation of purple photosynthetic bacteria. In: Blankenship RE, Madigan MT, Bauer C (eds) Anoxygenic photosynthetic bacteria. Kluwer Academic Publishers, Dordrecht, pp 1029–1065

    Google Scholar 

  • Wydrzynski TJ, Satoh K (eds) (2005) Photosystem II: the light-induced water:plastoquinone oxidoreductase. Springer, Dordrecht

    Google Scholar 

  • Yocum CF, Pecoraro VL (1999) Recent advances in the understanding of the biological chemistry of manganese. Curr Opin Chem Biol 3:182–187

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors thank Alicia Kishpaugh and Camille Black for their assistance in protein preparation. This study was supported by Grant CHE 1158552 from the National Science Foundation.

Author information

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ, 85287-1604, USA

    A. A. Tufts, M. Flores, T. L. Olson, J. C. Williams & J. P. Allen

Authors
  1. A. A. Tufts
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M. Flores
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. T. L. Olson
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. J. C. Williams
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. J. P. Allen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to J. P. Allen.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tufts, A.A., Flores, M., Olson, T.L. et al. Electronic structure of the Mn-cofactor of modified bacterial reaction centers measured by electron paramagnetic resonance and electron spin echo envelope modulation spectroscopies. Photosynth Res 120, 207–220 (2014). https://doi.org/10.1007/s11120-013-9887-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11120-013-9887-1

Keywords

Search

Navigation