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Pulsed Multifrequency Electron Paramagnetic Resonance Spectroscopy Reveals Key Branch Points for One- vs Two-Electron Reactivity in Mn/Fe Proteins.

ABSTRACT: Traditionally, the ferritin-like superfamily of proteins was thought to exclusively use a diiron active site in catalyzing a diverse array of oxygen-dependent reactions. In recent years, novel redox-active cofactors featuring heterobimetallic Mn/Fe active sites have been discovered in both the radical-generating R2 subunit of class Ic (R2c) ribonucleotide reductases (RNRs) and the related R2-like ligand-binding oxidases (R2lox). However, the protein-specific factors that differentiate the radical reactivity of R2c from the C-H activation reactions of R2lox remain unknown. In this work, multifrequency pulsed electron paramagnetic resonance (EPR) spectroscopy and ligand hyperfine techniques in conjunction with broken-symmetry density functional theory calculations are used to characterize the molecular and electronic structures of two EPR-active intermediates trapped during aerobic assembly of the R2lox Mn/Fe cofactor. A Mn^III(μ-O)(μ-OH)Fe^III species is identified as the first EPR-active species and represents a common state between the two classes of redox-active Mn/Fe proteins. The species downstream from the Mn^III(μ-O)(μ-OH)Fe^III state exhibits unique EPR properties, including unprecedented spectral breadth and isotope-dependent g-tensors, which are attributed to a weakly coupled, hydrogen-bonded Mn^III(μ-OH)Fe^III species. This final intermediate precedes formation of the Mn^III/Fe^III resting state and is suggested to be relevant to understanding the endogenous reactivity of R2lox.

SUBMITTER: Kisgeropoulos EC

PROVIDER: S-EPMC9433311 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

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Pulsed Multifrequency Electron Paramagnetic Resonance Spectroscopy Reveals Key Branch Points for One- vs Two-Electron Reactivity in Mn/Fe Proteins.

Kisgeropoulos Effie C EC Gan Yunqiao J YJ Greer Samuel M SM Hazel Joseph M JM Shafaat Hannah S HS

Journal of the American Chemical Society 20220705 27

Traditionally, the ferritin-like superfamily of proteins was thought to exclusively use a diiron active site in catalyzing a diverse array of oxygen-dependent reactions. In recent years, novel redox-active cofactors featuring heterobimetallic Mn/Fe active sites have been discovered in both the radical-generating R2 subunit of class Ic (R2c) ribonucleotide reductases (RNRs) and the related R2-like ligand-binding oxidases (R2lox). However, the protein-specific factors that differentiate the radica ...[more]

PMID: 35786920

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Project description:Spin-labeling and multifrequency EPR spectroscopy were used to probe the dynamic local structure of skeletal myosin in the region of force generation. Subfragment 1 (S1) of rabbit skeletal myosin was labeled with an iodoacetamide spin label at C707 (SH1). X- and W-band EPR spectra were recorded for the apo state and in the presence of ADP and nucleotide analogs. EPR spectra were analyzed in terms of spin-label rotational motion within myosin by fitting them with simulated spectra. Two models were considered: rapid-limit oscillation (spectrum-dependent on the orientational distribution only) and slow restricted motion (spectrum-dependent on the rotational correlation time and the orientational distribution). The global analysis of spectra obtained at two microwave frequencies (9.4 GHz and 94 GHz) produced clear support for the second model and enabled detailed determination of rates and amplitudes of rotational motion and resolution of multiple conformational states. The apo biochemical state is well-described by a single structural state of myosin (M) with very restricted slow motion of the spin label. The ADP-bound biochemical state of myosin also reveals a single structural state (M*, shown previously to be the same as the post-powerstroke ATP-bound state), with less restricted slow motion of the spin label. In contrast, the extra resolution available at 94 GHz reveals that the EPR spectrum of the S1.ADP.V(i)-bound biochemical state of myosin, which presumably mimics the S1.ADP.P(i) state, is resolved clearly into three spectral components (structural states). One state is indistinguishable from that of the ADP-bound state (M*) and is characterized by moderate restriction and slow motion, with a mole fraction of 16%. The remaining 84% (M**) contains two additional components and is characterized by fast rotation about the x axis of the spin label. After analyzing EPR spectra, myosin ATPase activity, and available structural information for myosin II, we conclude that post-powerstroke and pre-powerstroke structural states (M* and M**) coexist in the S1.ADP.V(i) biochemical state. We propose that the pre-powerstroke state M** is characterized by two structural states that could reflect flexibility between the converter and N-terminal domains of myosin.

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Dataset Information

Pulsed Multifrequency Electron Paramagnetic Resonance Spectroscopy Reveals Key Branch Points for One- vs Two-Electron Reactivity in Mn/Fe Proteins.

Publications

Pulsed Multifrequency Electron Paramagnetic Resonance Spectroscopy Reveals Key Branch Points for One- vs Two-Electron Reactivity in Mn/Fe Proteins.

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