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Leghemoglobin-derived radicals. Evidence for multiple protein-derived radicals and the initiation of peribacteroid
S Moreau1, M J Davies, C Mathieu
1Laboratoire de Biologie Végétale et Microbiologie, CNRS URA 1114, Université de Nice-Sophia Antipolis, 06108 Nice Cedex 2, France. m.davies@hri.edu.au
Abstract:
Reaction of H2O2 with ferric leghemoglobin (metLb, the monomeric, oxygen-carrying, heme protein from root nodules of nitrogen-fixing plants) has been previously shown to generate an iron(IV)-oxo (ferryl) species and at least one protein radical. The latter has been suggested to be a tyrosine-derived phenoxyl radical present at Tyr-133 in the soybean protein and Tyr-138 in the lupin protein. To obtain further information on these protein radicals and their potential interaction with the physiologically important peribacteroid membrane (which surrounds the microsymbiont in vivo), EPR spin trapping studies have been carried out with soybean metLb. Evidence has been obtained for at least two additional protein-derived radicals in addition to the phenoxyl radical; these radicals are transient and reactive in nature. These species are carbon-centered, and at least one is a tertiary species (.CR1R2R3); these radicals may be side chain- or alpha-carbon-derived, their exact sites have not been determined. Some of these radicals are on the protein surface and may be key intermediates in the formation of protein dimers. These radicals have been shown to be capable of reacting with peribacteroid membrane fractions, with the consequent generation of lipid-derived radicals. The formation of such radicals may result in the depletion of membrane antioxidants and the initiation of lipid peroxidation. This transfer of damage from the heme center via the protein surface to neighboring membranes may be of considerable biological significance; the destruction of this membrane is one of the earliest observable events in root nodule senescence and is associated with the loss of nitrogen-fixing activity.
Insights
Hydrogen peroxide reacts with ferric leghemoglobin (metHb) to form protein radicals. These radicals can damage nearby membranes, potentially impacting nitrogen fixation in plants.
Area of Science:
- Biochemistry
- Plant Biology
- Free Radical Chemistry
Background:
- Ferric leghemoglobin (metHb) reacts with H2O2 to form ferryl species and protein radicals.
- A tyrosine-derived phenoxyl radical at specific residues has been proposed.
- The interaction of these radicals with the peribacteroid membrane is not fully understood.
Purpose of the Study:
- Investigate protein radicals generated from soybean metHb using EPR spin trapping.
- Determine the nature and reactivity of these radicals.
- Assess their potential interaction with the peribacteroid membrane and its consequences.
Main Methods:
- Electron Paramagnetic Resonance (EPR) spin trapping studies.
- Utilized soybean ferric leghemoglobin (metHb).
- Incubated with hydrogen peroxide and analyzed radical species.
Main Results:
- Identified at least two additional, transient, carbon-centered protein radicals beyond the phenoxyl radical.
- Observed that some radicals are surface-exposed and may lead to protein dimerization.
- Demonstrated reaction of these radicals with peribacteroid membrane fractions, generating lipid radicals.
Conclusions:
- Protein radicals generated from metHb can initiate damage to the peribacteroid membrane.
- This damage involves lipid peroxidation and antioxidant depletion.
- Radical transfer from protein to membrane may contribute to root nodule senescence and loss of nitrogen fixation.