Abstract
Cryogenic radiolytic reduction is one of the most straightforward and convenient methods of generation and stabilization of reactive iron–oxygen intermediates for mechanistic studies in chemistry and biochemistry. The method is based on one-electron reduction of the precursor complex in frozen solution via exposure to the ionizing radiation at cryogenic temperatures. Such approach allows for accumulation of the fleeting reactive complexes which otherwise could not be generated at sufficient amount for structural and mechanistic studies. Application of this method allowed for characterizing of peroxo-ferric and hydroperoxo-ferric intermediates, which are common for the oxygen activation mechanism in cytochromes P450, heme oxygenases, and nitric oxide synthases, as well as for the peroxide metabolism by peroxidases and catalases.
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References
Sharonov YA (1986) The heme electronic structure of reduced cytochromes P 450 and P 420 as studied by low-temperature magnetic circular dichroism. Mol Biol (Moscow) 20:440–450
Solomon EI, Hanson MA (1999) Bioinorganic spectroscopy. In: Solomon EI, Lever ABP (eds) Inorganic electronic structure and spectroscopy, vol 2. Wiley, New York, pp 1–129
Keilin D, Hartree EF (1949) Effect of low temperature on the absorption spectra of hemoproteins, with observations on the absorption spectrum of oxygen. Nature 164:254–259
Strickland EH (1974) Aromatic contributions to circular dichroism spectra of proteins. CRC Crit Rev Biochem 2:113–175
Strickland EH, Horwitz J, Billups C (1969) Fine structure in the near-ultraviolet circular dichroism and absorption spectra of tryptophan derivatives and chymotrypsinogen A at 77 K. Biochemistry 8:3205–3213
Sharonov YA (1992) Substrate induced electronic-conformational interactions in active site of reduced bacterial cytochrome P 450CAM and analysis of the heme electronic structure. Mol Biol (Moscow) 26:1251–1262
Sharonov YA (2001) The energy level scheme for the ferryl heme in compound II of the peroxidase-catalase family as determined from analysis of low-temperature magnetic circular dichroism. Biochim Biophys Acta 1504:444–451
Solomon EI, Pavel EG, Loeb KE, Campochiaro C (1995) Magnetic circular dichroism spectroscopy as a probe of the geometric and electronic structure of non-heme ferrous enzymes. Coord Chem Rev 144:369–460
Honig B, Ebrey TG (1974) The structure and spectra of the chromophore of the visual pigments. Annu Rev Biophys Bioeng 3:151–177
Balashov SP, Ebrey TG (2001) Trapping and spectroscopic identification of the photointermediates of bacteriorhodopsin at low temperatures. Photochem Photobiol 73:453–462
Ponkratov VV, Friedrich J, Vanderkooi JM, Burin AL, Berlin YA (2004) Physics of proteins at low temperature. J Low Temp Phys 137:289–317
Frauenfelder H, Alberding NA, Ansari A, Braunstein D, Cowen BR, Hong MK, Iben IET, Johnson JB, Luck S et al (1990) Proteins and pressure. J Phys Chem 94:1024–1037
Miller LM, Chance MR (1995) Structural and electronic factors that influence oxygen affinities: A spectroscopic comparison of ferrous and cobaltous oxymyoglobin. Biochemistry 34:10170–10179
Nienhaus K, Lamb DC, Deng P, Nienhaus GU (2002) The effect of ligand dynamics on heme electronic transition band III in myoglobin. Biophys J 82:1059–1067
Tetreau C, Mouawad L, Murail S, Duchambon P, Blouquit Y, Lavalette D (2005) Disentangling ligand migration and heme pocket relaxation in cytochrome P450cam. Biophys J 88:1250–1263
Cupane A, Leone M, Vitrano E, Cordone L (1995) Low temperature optical absorption spectroscopy: an approach to the study of stereodynamic properties of hemeproteins. Eur Biophys J 23:385–398
Unno M, Chen H, Kusama S, Shaik S, Ikeda-Saito M (2007) Structural characterization of the fleeting ferric peroxo species in myoglobin: Experiment and theory. J Am Chem Soc 129:13394–13395
Beitlich T, Kuehnel K, Schulze-Briese C, Shoeman RL, Schlichting I (2007) Cryoradiolytic reduction of crystalline heme proteins: analysis by UV-vis spectroscopy and X-ray crystallography. J Synchrotron Radiat 14:11–23
Meyer B (1971) Low temperature spectroscopy. American Elsevier Publishing Co., New York, 653 pp
Douzou P (1977) Cryobiochemistry: an introduction. Academic, London, 286 pp
Sergeev GB, Batyuk VA (1981) Cryochemistry. Mir Publishers, Moscow, 298 pp
Franks F (1985) Biophysics and biochemistry at low temperatures. Cambridge University Press, Cambridge, 210 pp
Auld DS (1993) Low-temperature stopped-flow rapid-scanning spectroscopy: performance tests and use of aqueous salt cryosolvents. Meth Enzymol 226:553–565
Douzou P (1980) Cryoenzymology in aqueous media. Adv Enzymol Relat Areas Mol Biol 51:1–74
Douzou P, Balny C (1977) Cryoenzymology in mixed solvents without cosolvent effects on enzyme specific activity. Proc Natl Acad Sci U S A 74:2297–2300
Douzou P, Petsko GA (1984) Proteins at work: “stop-action” pictures at subzero temperatures. Adv Protein Chem 36:245–361
Daniel RM, Dunn RV, Finney JL, Smith JC (2003) The role of dynamics in enzyme activity. Annu Rev Biophys Biomol Struct 32:69–92
Bragger JM, Dunn RV, Daniel RM (2000) Enzyme activity down to −100 degrees C. Biochim Biophys Acta 1480:278–282
Douzou P, Hui Bon Hoa G, Maurel P, Travers F (1976) Physical chemical data for mixed solvents used in low temperature biochemistry. In: Fasman GD (ed) Physical and chemical data, vol 1, CRC handbook of biochemistry and molecular biology. CRC, Cleveland, pp 520–539
Wright WW, Guffanti GT, Vanderkooi JM (2003) Protein in sugar films and in glycerol/water as examined by infrared spectroscopy and by the fluorescence and phosphorescence of tryptophan. Biophys J 85:1980–1995
Nienhaus K, Nienhaus GU (2008) Ligand dynamics in heme proteins observed by Fourier transform infrared spectroscopy at cryogenic temperatures. Meth Enzymol 437:347–378
Fink AL, Cartwright SJ (1981) Cryoenzymology. CRC Crit Rev Biochem 11:145–207
Reat V, Finney JL, Steer A, Roberts MA, Smith J, Dunn R, Peterson M, Daniel R (2000) Cryosolvents useful for protein and enzyme studies below −100 degrees C. J Biochem Biophys Meth 42:97–103
Dashnau JL, Nucci NV, Sharp KA, Vanderkooi JM (2006) Hydrogen bonding and the cryoprotective properties of glycerol/water mixtures. J Phys Chem B 110:13670–13677
Douzou P (1973) Enzymology at sub-zero temperatures. Mol Cell Biochem 1:15–27
Douzou P (1977) Enzymology at subzero temperatures. Adv Enzymol Relat Areas Mol Biol 45:157–272
Cox RP (1978) Cryoenzymology: the use of fluid solvent mixtures at subzero temperatures for the study of biochemical reactions. Biochem Soc Trans 6:689–697
Fink AL (1986) Protein folding in cryosolvents and at subzero temperatures. Methods Enzymol 131:173–185
Privalov PL (1990) Cold denaturation of proteins. CRC Crit Rev Biochem Mol Biol 25:281–305
Prabhu NV, Sharp KA (2005) Heat capacity in proteins. Annu Rev Phys Chem 56:521–548
Larroque C, Maurel P, Balny C, Douzou P (1976) Practical potentiometric determinations of proton activity in hydro organic solvents at subzero temperatures. Anal Biochem 73:9–19
Orii Y, Morita M (1977) Measurement of the pH of frozen buffer solutions by using pH indicators. J Biochem Biophys Meth 81:163–168
Williams-Smith DL, Bray RC, Barber MJ, Tsopanakis AD, Vincent SP (1977) Changes in apparent pH on freezing aqueous buffer solutions and their relevance to biochemical electron-paramagnetic-resonance spectroscopy. Biochem J 167:593–600
Heger D, Klanova J, Klan P (2006) Enhanced protonation of cresol red in acidic aqueous solutions caused by freezing. J Phys Chem B 110:1277–1287
Schulze H, Ristau O, Jung C (1994) The proton activity at cryogenic temperatures–a possible influence on the spin state of the heme iron of cytochrome P-450cam in supercooled buffered solutions. Biochim Biophys Acta 1183:491–498
Sieracki NA, Hwang HJ, Lee MK, Garner DK, Lu Y (2008) A temperature independent pH (TIP) buffer for biomedical biophysical applications at low temperatures. Chem Commun: 823–825
Laidler KJ (1996) A glossary of terms used in chemical kinetics, including reaction dynamics. Pure Appl Chem 68:149–192
Denisov IG, Grinkova YV, Baas BJ, Sligar SG (2006) The ferrous-dioxygen intermediate in human cytochrome P450 3A4: Substrate dependence of formation and decay kinetics. J Biol Chem 281:23313–23318
Lefevre-Groboillot D, Boucher JL, Mansuy D, Stuehr DJ (2006) Reactivity of the heme-dioxygen complex of the inducible nitric oxide synthase in the presence of alternative substrates. FEBS J 273:180–191
Schlichting I, Berendzen J, Chu K, Stock AM, Maves SA, Benson DE, Sweet RM, Ringe D, Petsko GA, Sligar SG (2000) The catalytic pathway of cytochrome P450cam at atomic resolution. Science 287:1615–1622
Grinkova YV, Denisov IG, Waterman MR, Arase M, Kagawa N, Sligar SG (2008) The ferrous-oxy complex of human aromatase. Biochem Biophys Res Commun 372:379–382
Van Leeuwen JW, Butler J, Swallow AJ (1981) A non-equilibrium state of deoxyhaemoglobin. Temperature-dependence and oxygen binding. Biochim Biophys Acta 667:185–196
Sato F, Shiro Y, Sakaguchi Y, Iizuka T, Hayashi H (1990) Thermodynamic study of protein dynamic structure in the oxygen binding reaction of myoglobin. J Biol Chem 265:18823–18828
Filiaci M, Nienhaus GU (1997) The role of entropy in the discrimination between CO and O2 in myoglobin. Eur Biophys J 26:209–214
Tetreau C, Di Primo C, Lange R, Tourbez H, Lavalette D (1997) Dynamics of carbon monoxide binding with cytochromes P-450. Biochemistry 36:10262–10275
Barman T, Travers F, Balny C, Hui Bon Hoa G, Douzou P (1986) New trends in cryoenzymology: probing the functional role of protein dynamics by single-step kinetics. Biochimie 68:1041–1051
Denisov IG, Hung S-C, Weiss KE, Mclean MA, Shiro Y, Park S-Y, Champion PM, Sligar SG (2001) Characterization of the oxygenated intermediate of the thermophilic cytochrome P450 CYP119. J Inorg Biochem 87:215–226
Denisov IG, Ikeda-Saito M, Yoshida T, Sligar SG (2002) Cryogenic absorption spectra of hydroperoxo-ferric heme oxygenase, the active intermediate of enzymatic heme oxygenation. FEBS Lett 532:203–206
Denisov IG, Makris TM, Sligar SG (2002) Cryoradiolysis for the study of P450 reaction intermediates. Meth Enzymol 357:103–115
Spinks JWT, Woods RJ (1990) An introduction to radiation chemistry, 3rd edn. Wiley, New York, 574 pp
Woods RJ, Pikaev AK (1994) Applied radiation chemistry, radiation processing. Wiley, New York, 535 pp
Davydov R, Makris TM, Kofman V, Werst DE, Sligar SG, Hoffman BM (2001) Hydroxylation of camphor by reduced oxy-cytochrome P450cam: Mechanistic implications of EPR and ENDOR studies of catalytic intermediates in native and mutant enzymes. J Am Chem Soc 123:1403–1415
Denisov IG, Victoria DC, Sligar SG (2007) Cryoradiolytic reduction of heme proteins: Maximizing dose-dependent yield. Radiat Phys Chem 76:714–721
Davydov R, Kuprin S, Graeslund A, Ehrenberg A (1994) Electron paramagnetic resonance study of the mixed-valent diiron center in Escherichia coli ribonucleotide reductase produced by reduction of radical-free protein R2 at 77 K. J Am Chem Soc 116:11120–11128
Davydov R, Ledbetter-Rogers A, Martasek P, Larukhin M, Sono M, Dawson JH, Siler Masters BS, Hoffman BM (2002) EPR and ENDOR characterization of intermediates in the cryoreduced oxy-nitric oxide synthase heme domain with bound l-arginine or N-hydroxyarginine. Biochemistry 41:10375–10381
Davydov R, Kofman V, Fujii H, Yoshida T, Ikeda-Saito M, Hoffman BM (2002) Catalytic mechanism of heme oxygenase through EPR and ENDOR of cryoreduced oxy-heme oxygenase and its Asp 140 mutants. J Am Chem Soc 124:1798–1808
Garcia-Serres R, Davydov RM, Matsui T, Ikeda-Saito M, Hoffman BM, Huynh BH (2007) Distinct reaction pathways followed upon reduction of oxy-heme oxygenase and oxy-myoglobin as characterized by Mossbauer spectroscopy. J Am Chem Soc 129:1402–1412
Davydov R, Osborne RL, Kim SH, Dawson JH, Hoffman BM (2008) EPR and ENDOR studies of cryoreduced Compounds II of peroxidases and myoglobin. Proton-coupled electron transfer and protonation status of ferryl hemes. Biochemistry 47:5147–5155
Denisov IG, Mak PJ, Makris TM, Sligar SG, Kincaid JR (2008) Resonance Raman characterization of the peroxo and hydroperoxo intermediates in cytochrome P450. J Phys Chem A 112:13172–13179
Denisov IG, Makris TM, Sligar SG (2002) Formation and decay of hydroperoxo-ferric heme complex in horseradish peroxidase studied by cryoradiolysis. J Biol Chem 277:42706–42710
Mak PJ, Denisov IG, Victoria D, Makris TM, Deng T, Sligar SG, Kincaid JR (2007) Resonance Raman detection of the hydroperoxo intermediate in the cytochrome P450 enzymatic cycle. J Am Chem Soc 129:6382–6383
Mak PJ, Kincaid JR (2008) Resonance Raman spectroscopic studies of hydroperoxo derivatives of cobalt-substituted myoglobin. J Inorg Biochem 102:1952–1957
Davydov R, Kappl R, Huettermann J, Peterson JA (1991) EPR-spectroscopy of reduced oxyferrous-P450cam. FEBS Lett 295:113–115
Makris TM, Davydov R, Denisov IG, Hoffman BM, Sligar SG (2002) Mechanistic enzymology of oxygen activation by the cytochromes P450. Drug Metab Rev 34:691–708
Makris TM, Von Koenig K, Schlichting I, Sligar SG (2007) Alteration of P450 distal pocket solvent leads to impaired proton delivery and changes in heme geometry. Biochemistry 46:14129–14140
Gasyna Z (1979) Intermediate spin-states in one-electron reduction of oxygen-hemoprotein complexes at low temperature. FEBS Lett 106:213–218
Denisov IG, Makris TM, Sligar SG (2001) Cryotrapped reaction intermediates of cytochrome P450 studied by radiolytic reduction with phosphorus-32. J Biol Chem 276:11648–11652
Denisov IG, Dawson JH, Hager LP, Sligar SG (2007) The ferric-hydroperoxo complex of chloroperoxidase. Biochem Biophys Res Commun 363:954–958
Ibrahim M, Denisov IG, Makris TM, Kincaid JR, Sligar SG (2003) Resonance Raman spectroscopic studies of hydroperoxo-myoglobin at cryogenic temperatures. J Am Chem Soc 125:13714–13718
Ibrahim M, Kincaid JR (2004) Spectroscopic studies of peroxo/hydroperoxo derivatives of heme proteins and model compounds. J Porphyrins Phthalocyanines 8:215–225
Sligar SG, Makris TM, Denisov IG (2005) Thirty years of microbial P450 monooxygenase research: Peroxo-heme intermediates: the central bus station in heme oxygenase catalysis. Biochem Biophys Res Commun 338:346–354
Gantt SL, Denisov IG, Grinkova YV, Sligar SG (2009) The critical iron–oxygen intermediate in human aromatase. Biochem Biophys Res Commun 387:169–173
Schuler RH (1994) Three decades of spectroscopic studies of radiation produced intermediates. Radiat Phys Chem 43:417–423
Douzou P, Balny C (1978) Protein fractionation at subzero temperatures. Adv Protein Chem 32:77–189
Bonfils C, Saldana JL, Debey P, Maurel P, Balny C, Douzou P (1979) Fast photochemical reactions of cytochrome P450 at subzero temperatures. Biochimie 61:681–687
Douzou P (1982) Developments in low-temperature biochemistry and biology. Proc R Soc Lond B 217:1–28
Douzou P (1983) Cryoenzymology. Cryobiology 20:625–635
Daniel RM, Smith JC, Ferrand M, Hery S, Dunn R, Finney JL (1998) Enzyme activity below the dynamical transition at 220 K. Biophys J 75:2504–2507
Gasyna Z (1980) Unusual spin-state transitions in the reduction of ferrylmyoglobin at low temperature. Biochem Biophys Res Comm 93:637–644
Gasyna Z, Browett WR, Stillman MJ (1988) Low-temperature magnetic circular dichroism studies of the photoreaction of horseradish peroxidase compound I. Biochemistry 27:2503–2509
Browett WR, Gasyna Z, Stillman MJ (1988) Temperature dependence and electronic transition energies in the magnetic circular dichroism spectrum of horeseradish peroxidase compound I. J Am Chem Soc 110:3633–3640
Zollfrank J, Friedrich J, Vanderkooi JM, Fidy J (1991) Conformational relaxation of a low-temperature protein as probed by photochemical hole burning. Horseradish peroxidase. Biophys J 59:305–312
Manas ES, Vanderkooi JM, Sharp KA (1999) The effects of protein environment on the low temperature electronic spectroscopy of cytochrome c and microperoxidase-11. J Phys Chem 103B:6334–6348
Wright WW, Carlos Baez J, Vanderkooi JM (2002) Mixed trehalose/sucrose glasses used for protein incorporation as studied by infrared and optical spectroscopy. Anal Biochem 307:167–172
Khajehpour M, Rietveld I, Vinogradov S, Prabhu NV, Sharp KA, Vanderkooi JM (2003) Accessibility of oxygen with respect to the heme pocket in horseradish peroxidase. Proteins 53:656–666
Zelent B, Nucci NV, Vanderkooi JM (2004) Liquid and ice water and glycerol/water glasses compared by infrared spectroscopy from 295 to 12 K. J Phys Chem A 108:11141–11150
Nibbs J, Vinogradov SA, Vanderkooi JM, Zelent B (2004) Flexibility in proteins: tuning the sensitivity to O2 diffusion by varying the lifetime of a phosphorescent sensor in horseradish peroxidase. Photochem Photobiol 80:36–40
Ponkratov VV, Wiedersich J, Friedrich J, Vanderkooi JM (2007) Experiments with proteins at low temperature: what do we learn on properties in their functional state? J Chem Phys 126:16510–16514
Austin RH, Beeson KW, Eisenstein L, Frauenfelder H, Gunsalus IC (1975) Dynamics of ligand binding to myoglobin. Biochemistry 14:5355–5373
Frauenfelder H, Sligar SG, Wolynes PG (1991) The energy landscapes and motions of proteins. Science 254:1598–1603
Chen G, Fenimore PW, Frauenfelder H, Mezei F, Swenson J, Young RD (2008) Protein fluctuations explored by inelastic neutron scattering and dielectric relaxation spectroscopy. Phil Mag 88:3877–3883
Perrella M, Heyda A, Mosca A, Rossi-Bernardi L (1978) Isoelectric focusing and electrophoresis at subzero temperatures. Anal Biochem 88:212–224
Perrella M, Benazzi L, Cremonesi L, Vesely S, Viggiano G, Berger RL (1983) Subzero temperature quenching and electrophoretic methods for the isolation of protein reaction intermediates. J Biochem Biophys Meth 7:187–197
Perrella M, Denisov I (1995) Low-temperature electrophoresis methods. Meth Enzymol 259:468–487
Balny C, Le Peuch C, Debey P (1975) Low temperature column chromatography: application to microsomal hydroxylating system. Anal Biochem 63:321–330
Balny C, Debey P, Douzou P (1976) The sub-zero temperature chromatographic isolation of transient intermediates of a multi-step cycle: preparation of the substrate-free oxy-ferrous cytochrome P450. FEBS Lett 69:236–239
Debey P, Balny C, Douzou P (1973) Enzyme assay in microsomes below zero degrees. Proc Natl Acad Sci U S A 70:2633–2636
Acknowledgments
We gratefully acknowledge the contribution into development of these methods and collaboration with Drs. T.M. Makris, I. Schlichting, B.M. Hoffman, R.M. Davydov, M. Ikeda-Saito, J.R. Kincaid, and P.J. Mak, much of which resulted in the cited works. We appreciate the help provided by Dr. S. Toshkov at the Nuclear Radiation Lab, University of Illinois, Urbana-Champaign, and Dr. J. Bentley while using the 60Co source in the Notre Dame Radiation Laboratory (Notre Dame University, IN). Irradiations were conducted partly at the Notre Dame Radiation Laboratory, which is a facility of the U.S. Department of Energy, Office of Basic Energy Sciences. This work is supported by NIH grants GM31756 and GM33775 to S.G.S.
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Denisov, I.G., Grinkova, Y.V., Sligar, S.G. (2012). Cryoradiolysis and Cryospectroscopy for Studies of Heme-Oxygen Intermediates in Cytochromes P450. In: Bujalowski, W. (eds) Spectroscopic Methods of Analysis. Methods in Molecular Biology, vol 875. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-806-1_20
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