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Defining redox state of X-ray crystal structures by single-crystal ultraviolet visible microspectrophotometry
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
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2002 (English)In: Methods in Enzymology, ISSN 0076-6879, Vol. 353, 301-318 p.Article 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.

Place, publisher, year, edition, pages
2002. Vol. 353, 301-318 p.
National Category
Cell Biology
URN: urn:nbn:se:uu:diva-91906DOI: 10.1016/S0076-6879(02)53057-3ISI: 000176466500027OAI: oai:DiVA.org:uu-91906DiVA: diva2:164784
Available from: 2004-09-01 Created: 2004-09-01 Last updated: 2013-12-19Bibliographically approved
In thesis
1. Crystallography in Four Dimensions: Methods and Applications
Open this publication in new window or tab >>Crystallography in Four Dimensions: Methods and Applications
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. 50 p.
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 990
Molecular biophysics, horseradish peroxidase, redox reactions, X-ray crystallography, haem catalysis, molecular movies, radiation damage, Molekylär biofysik
National Category
urn:nbn:se:uu:diva-4301 (URN)91-554-5994-3 (ISBN)
Public defence
2004-09-22, Room B22, BMC, Husargatan 3, Uppsala, 13:00
Available from: 2004-09-01 Created: 2004-09-01 Last updated: 2013-03-15Bibliographically approved

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Carlsson, Gunilla H.Hajdu, Janos
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