X-ray damage to the Mn4Ca complex in single crystals of photosystem II: a case study for metalloprotein crystallography.

X-ray absorption spectroscopy was used to measure the damage caused by exposure to x-rays to the Mn(4)Ca active site in single crystals of photosystem II as a function of dose and energy of x-rays, temperature, and time. These studies reveal that the conditions used for structure determination by x-ray crystallography cause serious damage specifically to the metal-site structure. The x-ray absorption spectra show that the structure changes from one that is characteristic of a high-valent Mn(4)(III(2),IV(2)) oxo-bridged Mn(4)Ca cluster to that of Mn(II) in aqueous solution. This damage to the metal site occurs at a dose that is more than one order of magnitude lower than the dose that results in loss of diffractivity and is commonly considered safe for protein crystallography. These results establish quantitative x-ray dose parameters that are applicable to redox-active metalloproteins. This case study shows that a careful evaluation of the structural intactness of the active site(s) by spectroscopic techniques can validate structures derived from crystallography and that it can be a valuable complementary method before structure-function correlations of metalloproteins can be made on the basis of high-resolution x-ray crystal structures.

[1]  J Berendzen,et al.  The catalytic pathway of cytochrome p450cam at atomic resolution. , 2000, Science.

[2]  V. Yachandra,et al.  Orientation of calcium in the Mn4Ca cluster of the oxygen-evolving complex determined using polarized strontium EXAFS of photosystem II membranes. , 2004, Biochemistry.

[3]  J. Barber.  The photosystems: structure, function and molecular biology. , 1992 .

[4]  T. Ono,et al.  Structural Changes of D1 C-terminal α-Carboxylate during S-state Cycling in Photosynthetic Oxygen Evolution* , 2005, Journal of Biological Chemistry.

[5]  H. Dau,et al.  The tetra-manganese complex of photosystem II during its redox cycle - X-ray absorption results and mechanistic implications. , 2001, Biochimica et biophysica acta.

[6]  G. Dismukes,et al.  An evaluation of structural models for the photosynthetic water-oxidizing complex derived from spectroscopic and X-ray diffraction signatures , 2001, JBIC Journal of Biological Inorganic Chemistry.

[7]  Petra Fromme,et al.  Crystal structure of photosystem II from Synechococcus elongatus at 3.8 Å resolution , 2001, Nature.

[8]  U. Arndt.  Optimum X‐ray wavelength for protein crystallography , 1984 .

[9]  Elspeth Garman,et al.  'Cool' crystals: macromolecular cryocrystallography and radiation damage. , 2003, Current opinion in structural biology.

[10]  W. Burmeister,et al.  Structural changes in a cryo-cooled protein crystal owing to radiation damage. , 2000, Acta crystallographica. Section D, Biological crystallography.

[11]  R. Strange,et al.  An e.x.a.f.s. study of the manganese O2-evolving complex in purified Photosystem II membrane fractions. The S1 and S2 states. , 1992, The Biochemical journal.

[12]  Donald R. Ort,et al.  Oxygenic Photosynthesis: The Light Reactions , 1996, Advances in Photosynthesis and Respiration.

[13]  V. Yachandra,et al.  Manganese Cluster in Photosynthesis: Where Plants Oxidize Water to Dioxygen. , 1996, Chemical reviews.

[14]  L. Powers,et al.  Identifiction and assay of synchrotron radiation‐induced alterations on metalloenzymes and proteins , 1980, FEBS letters.

[15]  V. Yachandra,et al.  The water-oxidation complex in photosynthesis. , 2004, Biochimica et biophysica acta.

[16]  D. Adams,et al.  Incorporation of Fluoride into a Tetranuclear Mn/O/RCO2 Aggregate: Potential Relevance to Inhibition by Fluoride of Photosynthetic Water Oxidation , 1995 .

[17]  R. Debus,et al.  Evidence that the C-terminus of the D1 polypeptide of photosystem II is ligated to the manganese ion that undergoes oxidation during the S1 to S2 transition: an isotope-edited FTIR study. , 2004, Biochemistry.

[18]  V. DeRose,et al.  Where plants make oxygen: a structural model for the photosynthetic oxygen-evolving manganese cluster , 1993 .

[19]  H. Chu,et al.  Vibrational spectroscopy of the oxygen-evolving complex and of manganese model compounds. , 2001, Biochimica et biophysica acta.

[20]  R. D. Britt,et al.  55Mn ENDOR of the S2-State Multiline EPR Signal of Photosystem II: Implications on the Structure of the Tetranuclear Mn Cluster , 2000 .

[21]  Uwe Bergmann,et al.  The electronic structure of Mn in oxides, coordination complexes, and the oxygen-evolving complex of photosystem II studied by resonant inelastic X-ray scattering. , 2004, Journal of the American Chemical Society.

[22]  Guillem Aromí,et al.  Comparison of the Manganese Cluster in Oxygen-Evolving Photosystem II with Distorted Cubane Manganese Compounds through X-ray Absorption Spectroscopy. , 1999, Inorganic chemistry.

[23]  K. Sauer.  A role for manganese in oxygen evolution in photosynthesis , 1980 .

[24]  J. Penner‐Hahn,et al.  Reduced Derivatives of the Manganese Cluster in the Photosynthetic Oxygen-Evolving Complex , 1992 .

[25]  J. Messinger.  Evaluation of different mechanistic proposals for water oxidation in photosynthesis on the basis of Mn4OxCa structures for the catalytic site and spectroscopic data , 2004 .

[26]  U. Bergmann,et al.  Absence of Mn-centered oxidation in the S(2) --> S(3) transition: implications for the mechanism of photosynthetic water oxidation. , 2001, Journal of the American Chemical Society.

[27]  V. Legallais,et al.  Synchrotron X-ray studies of biological preparations at low temperatures with optical monitoring of sample integrity. , 1982, Analytical biochemistry.

[28]  James Barber,et al.  Architecture of the Photosynthetic Oxygen-Evolving Center , 2004, Science.

[29]  V. Yachandra,et al.  The Mn cluster in the S(0) state of the oxygen-evolving complex of photosystem II studied by EXAFS spectroscopy: are there three Di-mu-oxo-bridged Mn(2) moieties in the tetranuclear Mn complex? , 2002, Journal of the American Chemical Society.

[30]  O. Carugo,et al.  When X-rays modify the protein structure: radiation damage at work. , 2005, Trends in biochemical sciences.

[31]  C. Nave,et al.  Radiation damage to crystalline biological molecules: current view. , 2002, Journal of synchrotron radiation.

[32]  A. Zouni,et al.  Crystal Structure of Cyanobacterial Photosystem II at 3.2 A Resolution: A Closer Look at the Mn- Cluster , 2004 .

[33]  V. Yachandra.  Structure of the manganese complex in photosystem II: insights from X-ray spectroscopy. , 2002, Philosophical transactions of the Royal Society of London. Series B, Biological sciences.

[34]  M. Kusunoki,et al.  Spin-exchange interactions in the S2-state manganese tetramer in photosynthetic oxygen-evolving complex deduced from g=2 multiline EPR signal , 1999 .

[35]  A. Zouni,et al.  Purification, characterisation and crystallisation of photosystem II from Thermosynechococcus elongatus cultivated in a new type of photobioreactor. , 2005, Biochimica et biophysica acta.

[36]  Nobuo Kamiya,et al.  Crystal structure of oxygen-evolving photosystem II from Thermosynechococcus vulcanus at 3.7-Å resolution , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[37]  M. Chan,et al.  Support for a dimer of di-.mu.-oxo dimers model for the photosystem II manganese aggregate. Synthesis and properties of [(Mn2O2)2(tphpn)2](ClO4)4 , 1991 .

[38]  E. Garman,et al.  Physical and chemical considerations of damage induced in protein crystals by synchrotron radiation: a radiation chemical perspective. , 2002, Journal of synchrotron radiation.

[39]  R. Debus.  The manganese and calcium ions of photosynthetic oxygen evolution. , 1992, Biochimica et biophysica acta.

[40]  R. Ravelli,et al.  The 'fingerprint' that X-rays can leave on structures. , 2000, Structure.

[41]  Richard Henderson,et al.  Cryo-protection of protein crystals against radiation damage in electron and X-ray diffraction , 1990, Proceedings of the Royal Society of London. Series B: Biological Sciences.

[42]  M. Haumann,et al.  The structure of the manganese complex of Photosystem II in its dark-stable S1-state?EXAFS results in relation to recent crystallographic data , 2004 .

[43]  J L Sussman,et al.  Specific chemical and structural damage to proteins produced by synchrotron radiation. , 2000, Proceedings of the National Academy of Sciences of the United States of America.