uu.seUppsala University Publications
Change search
ReferencesLink to record
Permanent link

Direct link
The catalytic pathway of horseradish peroxidase at high resolution
Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology.
Show others and affiliations
2002 In: Nature, Vol. 417, 463-468 p.Article in journal (Refereed) Published
Place, publisher, year, edition, pages
2002. Vol. 417, 463-468 p.
URN: urn:nbn:se:uu:diva-91907OAI: oai:DiVA.org:uu-91907DiVA: diva2:164785
Available from: 2004-09-01 Created: 2004-09-01Bibliographically 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

Open Access in DiVA

No full text

By organisation
Department of Cell and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Total: 68 hits
ReferencesLink to record
Permanent link

Direct link