A W-Band Electron Nuclear Double Resonance Study of Single Crystals of <sup>14</sup>N and <sup>15</sup>N Azurin

Coremans, J. W. A.; Poluektov, Oleg G.; Groenen, E. J. J.; Canters, Gerard W.; Nar, Herbert; Messerschmidt, Albrecht

doi:10.1021/ja962076u

Cited by 79 publications

(138 citation statements)

References 41 publications

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“…Certainly one aspect of growth will involve technical developments. Development of multifrequency and time-domain approaches (6,8,38) will continue. Although it is our view that the vicinity of 35 GHz in general provides an optimum compromise between competing demands of spectral dispersion and sample handling, numerous biological problems do demand a higher frequency, and certain spectroscopic constraints will sometimes demand a lower frequency.…”

Section: Dioxygen Activation By Heme Enzymesmentioning

confidence: 99%

“…Although the samples used in ENDOR͞ESEEM studies of metalloenzymes almost always are frozen solutions (but see, for example, ref. 8), and thus contain a random distribution of all active-site orientations, we have shown that the desired information nonetheless can be obtained through analysis of a 2D dataset comprised of ''orientation-selective'' ENDOR spectra collected at fields (g values) across the EPR envelope of frozen-solution (5,9).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Electron-nuclear double resonance spectroscopy (and electron spin-echo envelope modulation spectroscopy) in bioinorganic chemistry

Hoffman

2003

Proc. Natl. Acad. Sci. U.S.A.

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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.T he invitation to write this article explained, ''Perspective articles are intended to survey, from the author's distinctive perspective, some frontier aspects of the featured field and to highlight important recent developments and challenges.'' Within that definition, I shall discuss the ways that advanced paramagnetic resonance techniques, namely electron-nuclear double resonance (ENDOR) spectroscopy (1) and its junior partner electron spin-echo envelope modulation (ESEEM) spectroscopy (2), help us understand how metal ions function in biological systems and provide information in all of the critical categories listed in Table 1. In these early days of proteomics, where the primary sequence of all proteins in an organism are knowable in principle, and increasingly in fact, and where large-scale efforts are under way to determine the resting-state x-ray crystal structures of these proteins, this article is a representative response to the broader question: what roles do spectroscopies play? The answer, of course, is many, and important ones. BackgroundKey information about the composition, bonding, and structure of a paramagnetic metal center can be obtained by analysis of the electron-nuclear hyperfine and nuclear quadrupole couplings (3) of nuclei associated with endogenous and exogenous metal ligands, enzymebound substrates, inhibitors, and products, as well as the metal ions themselves. In principle, these couplings can be derived from splittings observable in a center's EPR spectrum. For most metallo-biomolecules, however, a variety of factors make these interactions unresolvable for most (or any) nuclei, and the chemical information is lost.ENDOR and ESEEM spectroscopies (3-7) retrieve the missing information. They provide an NMR spectrum of those nuclei that interact with the electron spin of the paramagnetic center, and such spectra display orders-ofmagnitude-better resolution than the

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Section: Dioxygen Activation By Heme Enzymesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Electron-nuclear double resonance spectroscopy (and electron spin-echo envelope modulation spectroscopy) in bioinorganic chemistry

Hoffman

2003

Proc. Natl. Acad. Sci. U.S.A.

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“…The electronic structure of blue copper proteins has been studied by various quantum chemical techniques. 5,[21][22][23] Here we follow the choice of Gewirth and Solomon, 4 who used D 2d -symmetry to predict the signs of the MCD bands of plastocyanin. The z-axis of our molecular coordinate system is close to the Cu-Met direction, while the x-axis is along the Cu-Cys direction.…”

Section: Ligand Field Transitionsmentioning

confidence: 99%

Deconvolution and Assignment of Different Optical Transitions of the Blue Copper Protein Azurin from Optically Detected Electron Paramagnetic Resonance Spectroscopy

et al. 2001

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Abstract:Magnetic circular dichroism is a powerful spectroscopic tool for the assignment of optical resonance lines. An extension of this technique, microwave-modulated circular dichroism, provides additional details, in particular information about the orientation of optical transition moments. It arises from magnetization precessing around the static magnetic field, excited by a microwave field, in close analogy to electron paramagnetic resonance (EPR). In this paper we investigate the visible and near-infrared spectrum of the blue copper protein Pseudomonas aeruginosa azurin. Using a nonoriented sample (frozen solution), we apply this technique to measure the variation of the optical anisotropy with the wavelength. A comparison with the optical anisotropies of the possible ligand-field and charge-transfer transitions allows us to identify individual resonance lines in the strongly overlapping spectrum and assign them to specific electronic transitions. The technique is readily applicable to other proteins with transition metal centers.

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“…The magnitudes of the observed nitrogen couplings in center H are, however, in better agreement with those observed for histidine coordinated in N ␦ position to heme Fe III , and also histidine N ␦ ligating a quinone (60,62) which lie in a range of e 2 qQ/h ϭ 1.44 Ϫ 1.65 and ϭ 0.69 Ϫ 0.91. Values of e 2 qQ/h Ϸ 1.4 MHz and Ϸ 0.9 have been reported for the remote N of histidine coordinated to copper in azurin (63). However, the e 2 qQ/h values for 14 N of center H in R2-Y122H (1.6 and 1.3 MHz) are in remarkably good agreement with those reported for the histidine ligands in the structurally very similar diiron complexes of MMOH and semimethemerythrin sulfide (1.4 MHz), for which also comparable 14 N hf tensors were observed (64).…”

Section: Discussionmentioning

confidence: 84%

A New Tyrosyl Radical on Phe208 as Ligand to the Diiron Center in Escherichia coli Ribonucleotide Reductase, Mutant R2-Y122H

Kolberg

Logan

Bleifuß

et al. 2005

Journal of Biological Chemistry

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The R2 protein subunit of class I ribonucleotide reductase (RNR) belongs to a structurally related family of oxygen bridged diiron proteins. In wild-type R2 of Escherichia coli, reductive cleavage of molecular oxygen by the diferrous iron center generates a radical on a nearby tyrosine residue (Tyr 122 ), which is essential for the enzymatic activity of RNR, converting ribonucleotides into deoxyribonucleotides. In this work, we characterize the mutant E. coli protein R2-Y122H, where the radical site is substituted with a histidine residue. The x-ray structure verifies the mutation. R2-Y122H contains a novel stable paramagnetic center which we name H, and which we have previously proposed to be a diferric iron center with a strongly coupled radical, Fe is the only phenylalanine in the ligand sphere of the iron site, and generation of a phenyl radical requires a very high oxidation potential, we propose that in Y122H residue Phe 208 is hydroxylated, as observed earlier in another mutant (R2-Y122F/E238A), and further oxidized to a phenoxyl radical, which is coordinated to Fe1. This work demonstrates that small structural changes can redirect the reactivity of the diiron site, leading to oxygenation of a hydrocarbon, as observed in the structurally similar methane monoxygenase, and beyond, to formation of a stable iron-coordinated radical.Over the past decades a number of structurally related proteins that contain oxygen-bridged dinuclear iron centers have been discovered and characterized (1, 2). Among these are the hydroxylase protein component of methane monooxygenase (MMO), 1 called MMOH (3-5), and the R2 protein component of class I ribonucleotide reductase (RNR) (6 -8). These proteins reveal a strikingly similar coordination arrangement of their diiron centers, but fulfill entirely different biological functions; in MMOH, the diiron center is located directly at the substrate binding catalytic site and is directly responsible for the hydroxylation of methane or other substrates, whereas in R2, the diiron center is more than 35 Å away from the substrate binding active site located in the second protein subunit R1 (9 -11). The catalytic role of the diiron center in R2 is to generate and stabilize a tyrosyl radical (at position Tyr 122 in Escherichia coli R2), and this tyrosyl radical is connected to the active site in R1 via a chain of H-bonded amino acid residues. It has been proposed that upon substrate binding in R1 Tyr 122 transfers its radical character via this chain to a cysteine (Cys 439 in E. coli R1), which in turn starts substrate turnover from ribonucleotide to deoxyribonucleotide (9 -12).The R2 protein is a homodimer which in its active form contains two -oxo bridged diferric iron centers and a substoichiometric amount of a tyrosyl radical. The active form can be generated in vitro by the so-called reconstitution reaction by adding a 6-fold molar excess of Fe 2ϩ and molecular oxygen to the iron-free apoR2 protein (13). The tyrosyl radical is stabilized by a surrounding cluster of hydrophobic side ch...

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A W-Band Electron Nuclear Double Resonance Study of Single Crystals of ¹⁴N and ¹⁵N Azurin

Cited by 79 publications

References 41 publications

Electron-nuclear double resonance spectroscopy (and electron spin-echo envelope modulation spectroscopy) in bioinorganic chemistry

Electron-nuclear double resonance spectroscopy (and electron spin-echo envelope modulation spectroscopy) in bioinorganic chemistry

Deconvolution and Assignment of Different Optical Transitions of the Blue Copper Protein Azurin from Optically Detected Electron Paramagnetic Resonance Spectroscopy

A New Tyrosyl Radical on Phe208 as Ligand to the Diiron Center in Escherichia coli Ribonucleotide Reductase, Mutant R2-Y122H

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A W-Band Electron Nuclear Double Resonance Study of Single Crystals of 14N and 15N Azurin

Cited by 79 publications

References 41 publications

Electron-nuclear double resonance spectroscopy (and electron spin-echo envelope modulation spectroscopy) in bioinorganic chemistry

Electron-nuclear double resonance spectroscopy (and electron spin-echo envelope modulation spectroscopy) in bioinorganic chemistry

Deconvolution and Assignment of Different Optical Transitions of the Blue Copper Protein Azurin from Optically Detected Electron Paramagnetic Resonance Spectroscopy

A New Tyrosyl Radical on Phe208 as Ligand to the Diiron Center in Escherichia coli Ribonucleotide Reductase, Mutant R2-Y122H

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A W-Band Electron Nuclear Double Resonance Study of Single Crystals of ¹⁴N and ¹⁵N Azurin