Advanced paramagnetic resonance spectroscopies of iron–sulfur proteins: Electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM)

Abstract

The advanced electron paramagnetic resonance (EPR) techniques, electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) spectroscopies, provide unique insights into the structure, coordination chemistry, and biochemical mechanism of Nature’s widely distributed iron-sulfur cluster (FeS) proteins. This review describes the ENDOR and ESEEM techniques and then provides a series of case studies on their application to a wide variety of FeS proteins including ferredoxins, nitrogenase, and radical SAM enzymes.

Nitric Oxide Modulates Endonuclease III Redox Activity by a 800 mV Negative Shift upon [Fe4S4] Cluster Nitrosylation

“…46 The CW EPR spectrum of native EndoIII is devoid of any observable signal, consistent with a diamagnetic [Fe 4 S 4 ] 2+ cluster 47 , while nitrosylated EndoIII exhibits a rhombic signal centered at g = 2.029 consistent with the presence of an {Fe(NO) 2 } 9 dinitrosyl iron complex. 22,42 Interestingly, when nitrosylated EndoIII is reduced with dithionite, the {Fe(NO) 2 } 9 profile diminishes, and a new axial signal is observed with g ⊥ = 2.011 and g ∥ = 1.972.…”

Radical SAM catalysis via an organometallic intermediate with an Fe–[5′-C]-deoxyadenosyl bond

“…To first order, an ENDOR spectrum of an I = 1/2 nucleus (N) in a frozen solution comprises a superposition of signals from different orientations, each signal a doublet at frequencies, ν ± = |ν(N) ± A/2|, where ν(N) is the nuclear larmor frequency and A is the orientation-dependent hyperfine coupling. (23) For 13 C, A/2 ≪ ν( 13 C) and it is convenient to plot spectra vs ν - ν( 13 C). For 57 Fe, ν( 57 Fe) ≪ A/2 and spectra are plotted vs ν.…”

“…Upper : CW ENDOR spectra for 57 Fe-enriched (red) and natural abundance (gray) rfq samples. Lower : Frequency sweep and randomly hopped stochastic CW ENDOR spectra (23) for 57 Fe-enriched reduced PFL-AE. Conditions : microwave frequency = 35.45 GHz and 35.07GHz for rfq and 57 Fe-enriched reduced PFL-AE, respectively, microwave power = 1 mW, 100 kHz modulation amplitude = 1.3 G, rf sweep rate = 1 MHz/s, stochastic CW ENDOR cycle; rf-on = 3 ms, rf-off = 1 ms, sample collection time = 3 ms, and T = 2 K.…”

“…This radical initiator Fe-[5′-C]-adenosyl complex expands the growing list of enzymatic bio-organometallic centers, whose first entry was coenzyme B 12 ,(18, 19) but now includes the active-site metal clusters of hydrogenases,(22) nitrogenase,(23, 24) and a catalytic intermediate of IspH. (25, 26) Our study shows that radical SAM enzymes, the largest enzyme superfamily currently known, also can function through an organometallic center.…”

New NMR tools for protein structure and function: Spin tags for dynamic nuclear polarization solid state NMR

“…Studies of numerous proteins containing endogenous metals or radicals have illustrated the wealth of information that can be obtained through direct observation of the unpaired electron by EPR spectroscopy, observation of the paramagnetic tag's effect on the NMR spectrum of the bound protein [63], and hybrid methods studying hyperfine couplings in detail [64]. Labeling proteins with an exogenous radical tag was a natural extension of these methods to diamagnetic systems.…”