Out of plane distortions of the heme b of Escherichia coli succinate dehydrogenase

The role of the heme b in Escherichia coli succinate dehydrogenase is highly ambiguous and its role in catalysis is questionable. To examine whether heme reduction is an essential step of the catalytic mechanism, we generated a series of site-directed mutations around the heme binding pocket, creati...

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Bibliographic Details
Published in:PloS one 2012-02, Vol.7 (2), p.e32641
Main Authors: Tran, Quang M, Fong, Carmen, Rothery, Richard A, Maklashina, Elena, Cecchini, Gary, Weiner, Joel H
Format: Article
Language:English
Subjects:
Binding sites
Biochemistry
Biology
Biophysics
Catalysis
Catalytic activity
Cell Membrane - metabolism
Chemistry
Conformation
Cytochrome
Dehydrogenase
Dehydrogenases
Deoxyribonucleic acid
DNA
E coli
Electron paramagnetic resonance
Electron Spin Resonance Spectroscopy
Enzymes
Escherichia coli
Escherichia coli - enzymology
Heme
Heme - chemistry
Humans
Libraries
Medicine
Molecular biology
Molecular Conformation
Molecular structure
Mutagenesis
Mutagenesis, Site-Directed
Mutation
Oxidase
Oxidases
Oxidation
Oxidation-Reduction
Porphyrins - chemistry
Proteins
Reactive Oxygen Species
Redox potential
Redox properties
Reductase
Rhodospirillum
Spectrophotometry, Ultraviolet - methods
Succinate dehydrogenase
Succinate Dehydrogenase - chemistry
Sulfur
Superoxide
Superoxides
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Summary:The role of the heme b in Escherichia coli succinate dehydrogenase is highly ambiguous and its role in catalysis is questionable. To examine whether heme reduction is an essential step of the catalytic mechanism, we generated a series of site-directed mutations around the heme binding pocket, creating a library of variants with a stepwise decrease in the midpoint potential of the heme from the wild-type value of +20 mV down to -80 mV. This difference in midpoint potential is enough to alter the reactivity of the heme towards succinate and thus its redox state under turnover conditions. Our results show both the steady state succinate oxidase and fumarate reductase catalytic activity of the enzyme are not a function of the redox potential of the heme. As well, lower heme potential did not cause an increase in the rate of superoxide production both in vitro and in vivo. The electron paramagnetic resonance (EPR) spectrum of the heme in the wild-type enzyme is a combination of two distinct signals. We link EPR spectra to structure, showing that one of the signals likely arises from an out-of-plane distortion of the heme, a saddled conformation, while the second signal originates from a more planar orientation of the porphyrin ring.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0032641