Kinetic analysis of amino acid radicals formed in H2O2-driven CuILPMO reoxidation implicates dominant homolytic reactivity

Lytic polysaccharide monooxygenases (LPMOs) have been proposed to react with bothO2andH2O2as cosubstrates. In this study, theH2O2reaction with reducedHypocrea jecorinaLPMO9A (CuI-HjLPMO9A) is demonstrated to be 1,000-fold faster than theO2reaction while producing the same oxidized oligosaccharide products. Analysis of the reactivity in the absence of polysaccharide substrate by stopped-flow absorption and rapid freeze–quench (RFQ) electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) yields two intermediates corresponding to neutral tyrosyl and tryptophanyl radicals that are formed along minor reaction pathways. The dominant reaction pathway is characterized by RFQ EPR and kinetic modeling to directly produce CuII-HjLPMO9A and indicates homolytic O–O cleavage. Both optical intermediates exhibit magnetic exchange coupling with the CuIIsites reflecting facile electron transfer (ET) pathways, which may be protective against uncoupled turnover or provide an ET pathway to the active site with substrate bound. The reactivities of nonnative organic peroxide cosubstrates effectively exclude the possibility of a ping-pong mechanism.

Generation of guanine–amino acid cross-links by a free radical combination mechanism

The key step of DNA–protein cross-linking in vitro is the combination of guanine neutral radicals with side-chain C-centered amino acid radicals produced by a two-photon ionization method.

Pulsed electron–electron double resonance: beyond nanometre distance measurements on biomacromolecules

PELDOR (or DEER; pulsed electron–electron double resonance) is an EPR (electron paramagnetic resonance) method that measures via the dipolar electron–electron coupling distances in the nanometre range, currently 1.5–8 nm, with high precision and reliability. Depending on the quality of the data, the error can be as small as 0.1 nm. Beyond mere mean distances, PELDOR yields distance distributions, which provide access to conformational distributions and dynamics. It can also be used to count the number of monomers in a complex and allows determination of the orientations of spin centres with respect to each other. If, in addition to the dipolar through-space coupling, a through-bond exchange coupling mechanism contributes to the overall coupling both mechanisms can be separated and quantified. Over the last 10 years PELDOR has emerged as a powerful new biophysical method without size restriction to the biomolecule to be studied, and has been applied to a large variety of nucleic acids as well as proteins and protein complexes in solution or within membranes. Small nitroxide spin labels, paramagnetic metal ions, amino acid radicals or intrinsic clusters and cofactor radicals have been used as spin centres.

Long-range distance determinations in biomacromolecules by EPR spectroscopy

Electron paramagnetic resonance (EPR) spectroscopy provides a variety of tools to study structures and structural changes of large biomolecules or complexes thereof. In order to unravel secondary structure elements, domain arrangements or complex formation, continuous wave and pulsed EPR methods capable of measuring the magnetic dipole coupling between two unpaired electrons can be used to obtain long-range distance constraints on the nanometer scale. Such methods yield reliably and precisely distances of up to 80 Å, can be applied to biomolecules in aqueous buffer solutions or membranes, and are not size limited. They can be applied either at cryogenic or physiological temperatures and down to amounts of a few nanomoles. Spin centers may be metal ions, metal clusters, cofactor radicals, amino acid radicals, or spin labels. In this review, we discuss the advantages and limitations of the different EPR spectroscopic methods, briefly describe their theoretical background, and summarize important biological applications. The main focus of this article will be on pulsed EPR methods like pulsed electron–electron double resonance (PELDOR) and their applications to spin-labeled biosystems.