Electron--nuclear-double-resonance studies of spinach plastocyanin.
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ABSTRACT:
SUBMITTER: Yokoi H
PROVIDER: S-EPMC1152383 | biostudies-other | 1983 Oct
REPOSITORIES: biostudies-other
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Project description:The fleeting ferric peroxo and hydroperoxo intermediates of dioxygen activation by hemoproteins can be readily trapped and characterized during cryoradiolytic reduction of ferrous hemoprotein-O2 complexes at 77 K. Previous cryoannealing studies suggested that the relaxation of cryogenerated hydroperoxoferric intermediates of myoglobin (Mb), hemoglobin, and horseradish peroxidase (HRP), either trapped directly at 77 K or generated by cryoannealing of a trapped peroxo-ferric state, proceeds through dissociation of bound H2O2 and formation of the ferric heme without formation of the ferryl porphyrin π-cation radical intermediate, compound I (Cpd I). Herein we have reinvestigated the mechanism of decays of the cryogenerated hydroperoxyferric intermediates of α- and β-chains of human hemoglobin, HRP, and chloroperoxidase (CPO). The latter two proteins are well-known to form spectroscopically detectable quasistable Cpds I. Peroxoferric intermediates are trapped during 77 K cryoreduction of oxy Mb, α-chains, and β-chains of human hemoglobin and CPO. They convert into hydroperoxoferric intermediates during annealing at temperatures above 160 K. The hydroperoxoferric intermediate of HRP is trapped directly at 77 K. All studied hydroperoxoferric intermediates decay with measurable rates at temperatures above 170 K with appreciable solvent kinetic isotope effects. The hydroperoxoferric intermediate of β-chains converts to the S = 3/2 Cpd I, which in turn decays to an electron paramagnetic resonance (EPR)-silent product at temperature above 220 K. For all the other hemoproteins studied, cryoannealing of the hydroperoxo intermediate directly yields an EPR-silent majority product. In each case, a second follow-up 77 K γ-irradiation of the annealed samples yields low-spin EPR signals characteristic of cryoreduced ferrylheme (compound II, Cpd II). This indicates that in general the hydroperoxoferric intermediates relax to Cpd I during cryoanealing at low temperatures, but when this state is not captured by reaction with a bound substrate, it is reduced to Cpd II by redox-active products of radiolysis.
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Project description:The 5'-deoxyadenosyl radical (5'-dAdo·) abstracts a substrate H atom as the first step in radical-based transformations catalyzed by adenosylcobalamin-dependent and radical S-adenosyl-l-methionine (RS) enzymes. Notwithstanding its central biological role, 5'-dAdo· has eluded characterization despite efforts spanning more than a half-century. Here, we report generation of 5'-dAdo· in a RS enzyme active site at 12 K using a novel approach involving cryogenic photoinduced electron transfer from the [4Fe-4S]+ cluster to the coordinated S-adenosylmethionine (SAM) to induce homolytic S-C5' bond cleavage. We unequivocally reveal the structure of this long-sought radical species through the use of electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies with isotopic labeling, complemented by density-functional computations: a planar C5' (2pπ) radical (∼70% spin occupancy); the C5'(H)2 plane is rotated by ∼37° (experiment)/39° (DFT) relative to the C5'-C4'-(C4'-H) plane, placing a C5'-H antiperiplanar to the ribose-ring oxygen, which helps stabilize the radical against elimination of the 4'-H. The agreement between φ from experiment and in vacuo DFT indicates that the conformation is intrinsic to 5-dAdo· itself, and not determined by its environment.
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Project description:Proton electron nuclear double resonance (ENDOR) spectra from the iron-molybdenum cofactor (FeMoco) of Klebsiella pneumoniae nitrogenase bound to the enzyme show that a wide variety of substrates and inhibitors, including dinitrogen, acetylene and cyanide, do not bind at or close to FeMoco in the dithionite-reduced state of the free MoFe protein, in agreement with our previous kinetic studies. Therefore models for substrate binding to FeMoco must consider structures at a more reduced level than that described by Kim and Rees [(1992) Science 257, 1677-1682]. After the enzyme has turned over in the presence of 2H2O, an additional set of protons are potentially available for exchange, namely those that can give rise to dihydrogen during enzyme turnover or generate the hydridic dinitrogen binding site; such exchangeable protons were not observed. They cannot therefore be proposed in order to explain the unusual geometry of the 'trigonal iron atoms' observed in the structure of FeMoco.
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Project description: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. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.
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Project description:This perspective discusses the ways that advanced paramagnetic resonance techniques, namely electron-nuclear double resonance (ENDOR) and electron spin-echo envelope modulation (ESEEM) spectroscopies, can help us understand how metal ions function in biological systems.
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Project description:Electron nuclear double-resonance ('ENDOR') spectroscopic studies on pig plasma amine oxidase have been carried out at 15 K. Deuterium-exchange studies show the presence of two sets of exchangeable protons, probably from two water molecules; from the magnitude of their hyperfine couplings, one is assigned to be equatorially, and the other axially, co-ordinated. Only one 14N hyperfine coupling is observed, suggesting that the bonding of all amino acid (histidine) or organic cofactor ligands is similar. Upon addition of azide, a further hyperfine coupling to nitrogen is observed which is smaller than that observed for the native enzyme; the hyperfine couplings to the remaining nitrogens are slightly altered.
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Project description:Crystallographic studies have shown that the F429H mutation of cytochrome P450 2B4 introduces an H-bond between His429 and the proximal thiolate ligand, Cys436, without altering the protein fold but sharply decreases the enzymatic activity and stabilizes the oxyferrous P450 2B4 complex. To characterize the influence of this hydrogen bond on the states of the catalytic cycle, we have used radiolytic cryoreduction combined with electron paramagnetic resonance (EPR) and (electron-nuclear double resonance (ENDOR) spectroscopy to study and compare their characteristics for wild-type (WT) P450 2B4 and the F429H mutant. (i) The addition of an H-bond to the axial Cys436 thiolate significantly changes the EPR signals of both low-spin and high-spin heme-iron(III) and the hyperfine couplings of the heme-pyrrole (14)N but has relatively little effect on the (1)H ENDOR spectra of the water ligand in the six-coordinate low-spin ferriheme state. These changes indicate that the H-bond introduced between His and the proximal cysteine decreases the extent of S ? Fe electron donation and weakens the Fe(III)-S bond. (ii) The added H-bond changes the primary product of cryoreduction of the Fe(II) enzyme, which is trapped in the conformation of the parent Fe(II) state. In the wild-type enzyme, the added electron localizes on the porphyrin, generating an S = (3)/2 state with the anion radical exchange-coupled to the Fe(II). In the mutant, it localizes on the iron, generating an S = (1)/2 Fe(I) state. (iii) The additional H-bond has little effect on g values and (1)H-(14)N hyperfine couplings of the cryogenerated, ferric hydroperoxo intermediate but noticeably slows its decay during cryoannealing. (iv) In both the WT and the mutant enzyme, this decay shows a significant solvent kinetic isotope effect, indicating that the decay reflects a proton-assisted conversion to Compound I (Cpd I). (v) We confirm that Cpd I formed during the annealing of the cryogenerated hydroperoxy intermediate and that it is the active hydroxylating species in both WT P450 2B4 and the F429H mutant. (vi) Our data also indicate that the added H-bond of the mutation diminishes the reactivity of Cpd I.
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Project description:The recent introduction of shaped pulses to Double Electron Electron Resonance (DEER) spectroscopy has led to significant enhancements in sensitivity through increased excitation bandwidths and improved control over spin dynamics. The application of DEER has so far relied on the presence of an incoherent pump channel to average out most undesired coherent effects of the pump pulse(s) on the observer spins. However, in fully coherent EPR spectrometers that are increasingly used to generate shaped pulses, the presence of coherent pump pulses means that these effects need to be explicitly considered. In this paper, we examine the effects of coherent rectangular and sech/tanh pump pulses in DEER experiments with up to three pump pulses. We show that, even in the absence of significant overlap of the observer and pump pulse excitation bandwidths, coherence transfer pathways involving both types of pulses generate spin echoes of considerable intensity. These echoes introduce artefacts, which, if not identified and removed, can easily lead to misinterpretation. We demonstrate that the observed echoes can be quantitatively modelled using a simple spin quantum dynamics approach that includes instrumental transfer functions. Based on an analysis of the echo crossing artefacts, we propose efficient phase cycling schemes for their suppression. This enables the use of advanced DEER experiments, characterized by high sensitivity and increased accuracy for long-distance measurements, on novel fully coherent EPR spectrometers.
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Project description:We present electron paramagnetic resonance experiments for which solid effect dynamic nuclear polarization transitions were observed indirectly via polarization loss on the electron. This use of indirect observation allows characterization of the dynamic nuclear polarization (DNP) process close to the electron. Frequency profiles of the electron-detected solid effect obtained using trityl radical showed intense saturation of the electron at the usual solid effect condition, which involves a single electron and nucleus. However, higher order solid effect transitions involving two, three, or four nuclei were also observed with surprising intensity, although these transitions did not lead to bulk nuclear polarization--suggesting that higher order transitions are important primarily in the transfer of polarization to nuclei nearby the electron. Similar results were obtained for the SA-BDPA radical where strong electron-nuclear couplings produced splittings in the spectrum of the indirectly observed solid effect conditions. Observation of high order solid effect transitions supports recent studies of the solid effect, and suggests that a multi-spin solid effect mechanism may play a major role in polarization transfer via DNP.
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Project description:The molybdenum centre of spinach (Spinacia oleracea) nitrate reductase has been investigated by e.p.r. spectroscopy of molybdenum(V) in reduced forms of the enzyme. The resting enzyme gives no signals attributable to Mo(V). However, on reduction with NADH, Mo(V) signals appeared at relatively short reaction times but decreased again on prolonged exposure to excess of the substrate as the enzyme was further reduced. On brief treatment of such samples with nitrate, Mo(V) signals reappeared but disappeared again on longer exposure to excess nitrate as the enzyme became fully reoxidized. Detailed investigation of the signals carried out in both 1H2O and 2H2O revealed the presence of two signal-giving species, referred to as 'signal A' and 'signal B', analogous to corresponding signals from nitrate reductase from Escherichia coli and from liver sulphite oxidase. Signal A has gav. 1.9767 and shows coupling to a single proton, exchangeable with the solvent, with A(1H)av. 1.3mT, whereas signal B shows no more than weak coupling to protons. Investigation of interconversion between the two species indicated that decreasing the pH from 8.0 to 6.7 had little effect, but that signal A was favoured by the presence of Cl-. This suggests, by analogy with recent work on sulphite oxidase by Bray, Gutteridge, Lamy & Wilkinson [Biochem. J. (1983) 211, 227-236] that Cl- is a ligand of molybdenum in the species giving signal A.
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