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Crystallography in Four Dimensions: Methods and Applications
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The four-electron reduction of dioxygen to water is the most exothermic non-photochemical reaction available to biology. A detailed molecular description of this reaction is needed to understand oxygen-based redox processes. Horseradish peroxidase (HRP) is a haem-containing redox enzyme capable of catalysing the reduction of dioxygen to water. We developed instrumentation and experimental methodology to capture and characterise by X-ray crystallography transient reaction intermediates in this reaction.

An instrument was designed (“the vapour stream system”) to facilitate reaction initiation, monitoring and intermediate trapping. In combination with single crystal microspectrophotometry, it was used to obtain conditions for capturing a reactive dioxygen complex in HRP. X-ray studies on oxidised intermediates can be difficult for various reasons. Electrons re-distributed in the sample through the photoelectric effect during X-ray exposure can react with high-valency intermediates. In order to control such side reactions during data collection, we developed a new method based on an angle-resolved spreading of the X-ray dose over many identical crystals. Composite data sets built up from small chunks of data represent crystal structures which received different X-ray doses. As the number of electrons liberated in the crystal is dose dependent, this method allows us to observe and drive redox reactions electron-by-electron in the crystal, using X-rays.

The methods developed here were used to obtain a three-dimensional movie on the X-ray-driven reduction of dioxygen to water in HRP. Separate experiments established high resolution crystal structures for all intermediates, showing such structures with confirmed redox states for the first time.

Activity of HRP is influenced by small molecule ligands, and we also determined the structures of HRP in complex with formate, acetate and carbon monoxide.

Other studies established conditions for successfully trapping the M-intermediate in crystals of mutant bacteriorhodopsin, but the poor diffraction quality of these crystals prevented high-resolution structural studies.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2004. , p. 50
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 990
Keyword [en]
Molecular biophysics, horseradish peroxidase, redox reactions, X-ray crystallography, haem catalysis, molecular movies, radiation damage
Keyword [sv]
Molekylär biofysik
National Category
Biophysics
Identifiers
URN: urn:nbn:se:uu:diva-4301ISBN: 91-554-5994-3 (print)OAI: oai:DiVA.org:uu-4301DiVA, id: diva2:164787
Public defence
2004-09-22, Room B22, BMC, Husargatan 3, Uppsala, 13:00
Opponent
Supervisors
Available from: 2004-09-01 Created: 2004-09-01 Last updated: 2013-03-15Bibliographically approved
List of papers
1. Protein crystallography in a vapour steam: data collection, reaction initiation and intermediate trapping in naked hydrated protein crystals
Open this publication in new window or tab >>Protein crystallography in a vapour steam: data collection, reaction initiation and intermediate trapping in naked hydrated protein crystals
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2002 (English)In: Journal of applied crystallography, ISSN 0021-8898, E-ISSN 1600-5767, Vol. 35, no 1, p. 113-116Article in journal (Refereed) Published
Abstract [en]

A procedure is presented for experiments on naked unfrozen protein crystals with the crystal mounted in a conventional cryo-loop and surrounded by a stream of a wet gas. The composition and temperature of the vapour stream can be adjusted to keep the crystal without deterioration for many hours. The arrangement allows (i) for rapidly testing crystals for diffraction before freezing, (ii) for data collection between 268-303 K with greatly reduced background, (iii) for the controlled drying or wetting of crystals, (iv) for the anaerobic manipulation of protein crystals, and (v) for the introduction of gaseous or volatile ingredients and reactants into the crystal. The technique offers new experimental possibilities, e.g. in time-resolved structural studies. Reaction initiation in many protein crystals can be achieved by changing the composition of the vapour stream to create a new chemical environment around the crystal and to introduce substrates/reactants either in the gas phase or as microdroplets. Spectral changes during such reactions can be monitored by single-crystal microspectrophotometry, and, once an intermediate has been detected at high concentrations, the crystal can be frozen, e.g. by rapidly switching the warm vapour stream to a cryogenically cooled helium or nitrogen jet. Representative examples are presented in this paper.

National Category
Medical and Health Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-91905 (URN)10.1107/S0021889801020702 (DOI)
Available from: 2004-09-01 Created: 2004-09-01 Last updated: 2017-12-14Bibliographically approved
2. Defining redox state of X-ray crystal structures by single-crystal ultraviolet visible microspectrophotometry
Open this publication in new window or tab >>Defining redox state of X-ray crystal structures by single-crystal ultraviolet visible microspectrophotometry
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2002 (English)In: Methods in Enzymology, ISSN 0076-6879, E-ISSN 1557-7988, Vol. 353, p. 301-318Article in journal (Refereed) Published
Abstract [en]

Exciting results have been emerging from the field of single-crystal X-ray crystallography, giving unprecedented detail of freeze-trapped reaction intermediates from important classes of macromolecules that contain chromophores. These structures have been coupled with single-crystal UV-visible microspectrophotometry. This has defined the distinct catalytic intermediates present in the crystal structures, allowing the correlation of electronic transitions with the observed structural transitions. Of particular note is that many of these structures have been generated “on the fly” during kinetic turnover in the crystal. Most enzymatic reactions proceed through distinct catalytic intermediates that, under favorable conditions, may accumulate transiently in the crystal during turnover. In some cases, the physical constraints of the contacts within crystals may also lead to a significant slowing of the reaction at certain points along the pathway where conformational changes are required. This can lead to a transient build-up of spectrally distinct intermediates in the crystal that can be trapped by flash freezing in liquid nitrogen, allowing a complete single-crystal data set to be collected to the highest possible resolution at a later time. Similar build-up of intermediates may be achieved by altering the pH, temperature, or the solvent environment around the protein in the crystal, or by producing engineered variants that build up an intermediate of interest. The chapter focuses on the technical considerations required to carry out UV-visible microspectroscopy of single crystals.

National Category
Cell Biology
Identifiers
urn:nbn:se:uu:diva-91906 (URN)10.1016/S0076-6879(02)53057-3 (DOI)000176466500027 ()
Available from: 2004-09-01 Created: 2004-09-01 Last updated: 2017-12-14Bibliographically approved
3. The catalytic pathway of horseradish peroxidase at high resolution
Open this publication in new window or tab >>The catalytic pathway of horseradish peroxidase at high resolution
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2002 In: Nature, Vol. 417, p. 463-468Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-91907 (URN)
Available from: 2004-09-01 Created: 2004-09-01Bibliographically approved
4. Complexes of horseradish peroxidase with formate, acetate and carbon monoxide
Open this publication in new window or tab >>Complexes of horseradish peroxidase with formate, acetate and carbon monoxide
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2005 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, no 2, p. 635-642Article in journal (Refereed) Published
Abstract [en]

Carbon monoxide, formate, and acetate interact with horseradish peroxidase (HRP) by binding to subsites within the active site. These ligands also bind to catalases, but their interactions are different in the two types of enzymes. Formate (notionally the “hydrated” form of carbon monoxide) is oxidized to carbon dioxide by compound I in catalase, while no such reaction is reported to occur in HRP, and the CO complex of ferrocatalase can only be obtained indirectly. Here we describe high-resolution crystal structures for HRP in its complexes with carbon monoxide and with formate, and compare these with the previously determined HRP−acetate structure [Berglund, G. I., et al. (2002) Nature 417, 463−468]. A multicrystal X-ray data collection strategy preserved the correct oxidation state of the iron during the experiments. Absorption spectra of the crystals and electron paramagnetic resonance data for the acetate and formate complexes in solution correlate electronic states with the structural results. Formate in ferric HRP and CO in ferrous HRP bind directly to the heme iron with iron−ligand distances of 2.3 and 1.8 Å, respectively. CO does not bind to the ferric iron in the crystal. Acetate bound to ferric HRP stacks parallel with the heme plane with its carboxylate group 3.6 Å from the heme iron, and without an intervening solvent molecule between the iron and acetate. The positions of the oxygen atoms in the bound ligands outline a potential access route for hydrogen peroxide to the iron. We propose that interactions in this channel ensure deprotonation of the proximal oxygen before binding to the heme iron.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-91908 (URN)10.1021/bi0483211 (DOI)
Available from: 2004-09-01 Created: 2004-09-01 Last updated: 2017-12-14Bibliographically approved

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