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Complexes of horseradish peroxidase with formate, acetate and carbon monoxide
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
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2005 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, no 2, 635-642 p.Article 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.

Place, publisher, year, edition, pages
2005. Vol. 44, no 2, 635-642 p.
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-91908DOI: 10.1021/bi0483211OAI: oai:DiVA.org:uu-91908DiVA: diva2:164786
Available from: 2004-09-01 Created: 2004-09-01 Last updated: 2017-12-14Bibliographically 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.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 990
Keyword
Molecular biophysics, horseradish peroxidase, redox reactions, X-ray crystallography, haem catalysis, molecular movies, radiation damage, Molekylär biofysik
National Category
Biophysics
Identifiers
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
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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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