Structural analysis of botulinum neurotoxin types A and E in aqueous and nonpolar solvents by Fourier transform infrared, second derivative UV absorption, and circular dichroic spectroscopies

Two pharmacologically similar but antigenically distinct botulinum neurotoxins, types A and E with a 1000-fold difference in their toxicity, were examined for nonpolar solvent-induced changes in secondary structures and polypeptide foldings to understand their structural differences and their comparative responsiveness/susceptibility to solvent perturbation. Analysis of far UV circular dichroic spectra in aqueous buffer for types A and E neurotoxins yielded the following: the -helix contents were 27 and 20%; the -sheets were 36 and 44%, the -turns were 6.0 and 0%, and the random coils were 31 and 36%, respectively. Fourier transform infrared spectra, obtained by using attenuated total reflection technique, indicated high content of -helix and -pleated sheet structures for both neurotoxins as judged by strong bands at 1651 and 1633 cm^–1 in the amide I frequency region and bands at 1314 and 1245 cm^–1 in the amide III frequency region. The peak height ratio of 1314 and 1245 cm^–1 bands, suggests that the type A neurotoxin has slightly higher -helical content than the type E neurotoxin. These observations are consistent with the secondary structures estimated from far UV circular dichroic spectra. Fourier transform infrared spectra of the neurotoxins, exposed to methanol, showed sharp increases of the 1651 cm^–1 band and a significant increase in the height of the 1314 cm^–1 band, suggesting increases in the -helical contents of the proteins. The changes were more in the type A than in the type E neurotoxin. The changes were reversible upon reexposure of the proteins to the aqueous buffer. Second derivative absorption spectroscopy demonstrated that methanol also induced changes in the degree of Tyr exposure to solvent. The results are discussed in terms of structural differences between the single and dichain neurotoxins and in terms of their mode of action.     

Comparative analysis of the amino- and carboxy-terminal domains of calmodulin by Fourier transform infrared spectroscopy

Fourier transform infrared spectra were obtained for mammalian calmodulin and two of its fragments produced by limited proteolysis with trypsin TR₁C (1–77) and TR₂C (78–148). Experiments were done in H₂O, D₂O and D₂O/trifluoroethanol (TFE) mixtures. Information about secondary structure was obtained from analysis of the amide I and II bands; while characteristic absorbances for tyrosine, phenylalanine and carboxylate groups were analyzed for changes in tertiary structure. Our data indicate that the secondary and tertiary structure is preserved in the two half molecules of CaM, both in the apo- and Ca²⁺-saturated state. Addition of the structure-inducing solvent TFE causes marked changes only in the apo-TR₁C domain. The maximum wavenumber for the amide I band of the two domains of CaM in D20 was markedly different (1642 cm^–1 for TR₁C versus 1646/1648 cm^–1 for Ca ²⁺ and apo-TR₂C). This renders the amide I band for the intact protein very broad in comparison to that in other proteins and is indicative of a distribution of -helices with slightly different hydrogen bonding patterns.     

Determination of secondary structure of normal fibrin from human peripheral blood

The secondary structure of human fibrin from normal donors and from bovine and suilline plasma was studied by Fourier transform ir spectroscopy and a quantitative analysis of its secondary structure was suggested. For this purpose, a previously experimented spectrum deconvolution procedure based on the use of the Conjugate Gradient Minimisation Algorithm with the addition of suitable constraints was applied to the analysis of conformation-sensitive amide bands. This procedure was applied to amide I and III analysis of bovine and suilline fibrin, obtained industrially, and to amide III analysis of human fibrin clots. The analysis of both amide I and III in the first case was useful in order to test the reliability of the method. We found bovine, suilline, and human fibrin to contain about 30% α-helix (amide I and III components at 1653 cm⁻¹, and 1312 and 1284 cm⁻¹, respectively), 40% β-sheets (amide I and III components at 1625 and 1231 cm⁻¹, respectively) and 30% turns (amide I and III components at 1696, 1680, 1675 cm⁻¹, and 1249 cm⁻¹, respectively). The precision of the quantitative determination depends on the amount of these structures in the protein. Particularly, the coefficient of variation is < 10% for percentage values of amide I and III components > 15 and 5%, respectively. The good agreement of our quantitative data, obtained separately by amide I and amide III analysis, and consistent with a previous fibrinogen (from commercial sources) study that reports only information about fibrin β-sheet content obtained by factor analysis, leads us to believe that the amounts of secondary structures found (α-helix, β-sheets, and turns) are accurate. © 1997 John Wiley & Sons, Inc. Biopoly 41: 545–553, 1997.     

FTIR spectroscopy of primary donor photooxidation in Photosystem I, Heliobacillus mobilis, and Chlorobium limicola. Comparison with purple bacteria

The photooxidation of the primary electron donor in several Photosystem I-related organisms (Synechocystis sp. PCC 6803, Heliobacillus mobilis, and Chlorobium limicola f. sp. thiosulphatophilum) has been studied by light-induced FTIR difference spectroscopy at 100 K in the 4000 to 1200 cm^–1 spectral range. The data are compared to the well-characterized FTIR difference spectra of the photooxidation of the primary donor P in Rhodobacter sphaeroides (both wild type and the heterodimer mutant HL M202) in order to get information on the charge localization and the extent of coupling within the (bacterio)chlorophylls constituting the oxidized primary donors. In Rb. sphaeroides RC, four marker bands mostly related to the dimeric nature of the oxidized primary donor have been previously observed at 2600, 1550, 1480, and 1295 cm^–1. The high-frequency band has been shown to correspond to an electronic transition (Breton et al. (1992) Biochemistry 31: 7503–7510) while the three other marker bands have been described as phase-phonon bands (Reimers and Hush (1995) Chem Phys 197: 323–332). The absence of these bands in PS I as well as in the heterodimer HL M202 demonstrates that in P700⁺ the charge is essentially localized on a single chlorophyll molecule. For both H. mobilis and C. limicola, the presence of a high-frequency band at 2050 and 2450 cm^–1, respectively, and of phase-phonon bands (at 1535 and 1300 cm^–1 in H. mobilis, at 1465 and 1280 cm^-1 in C. limicola) indicate that the positive charge in the photooxidized primary donor is shared between two coupled BChls. The structure of P840⁺ in C. limicola, in terms of the resonance interactions between the two BChl a molecules constituting the oxidized primary donor, is close to that of P⁺ in purple bacteria reaction centers while for H. mobilis the FTIR data are interpreted in terms of a weaker coupling of the two bacteriochlorophylls.Abbreviations (B)Chl (bacterio)chlorophyll - BPhe bacteriopheophytin - C. Chlorobium - FTIR Fourier transform infrared - H. Heliobacillus - PS I, PS II Photosystem I, Photosystem II - P primary electron donor - RC reaction center - Rb. Rhodobacter - Rp. Rhodopseudomonas - Q_A primary quinone acceptor - Wt wild type     

Step-scan FTIR spectroscopy resolves the QA −QB → QAQB − transition in Rb. sphaeroides R26 reaction centres

It is shown that step-scan Fourier transform infrared spectroscopy can be applied to resolve the Q_A ^–Q_B Q_AQ_B ^– transition in Rhodobacter sphaeroides reaction centres with a 5 µs time resolution. In the mid-infrared region (1900 – 1200 cm^–1), transient signals previously assigned to Q_A/B and Q_A/B ^– vibrations, respectively (Brudler et al. 1994; Brudler et al. 1995; Breton and Nabedryk 1996), can be resolved with this new technique. In addition, the three small positive bands in the spectral region of the carboxylic C=O stretching modes of acidic amino acid side chains are also resolved at 1730, 1719 and 1704 cm^–1. A global fit analysis yields two exponentials with half-times of 150 µs and 1.2 ms in agreement with IR spectroscopic studies at single wavenumbers (Hienerwadel et al. 1995), in the UV/VIS and near IR (Tiede et al. 1996, Li et al. 1996). The establishement of the step-scan technique enables a new approach to elucidate the molecular mechanism of this transition.     

Tetanus toxin conformation

Summary The circular dichroic spectrum of highly purified tetanus toxin has been determined between 200–310 nm. A comparison of the ellipticity between 207–243 nm and of the rotational strengths of the major resolved bands between 200–250 nm with the corresponding values from proteins of known conformation indicates that tetanus toxin contains about 20% -helix and 23% -structure. Above 250 nm the resolved spectrum showed contributions from tryptophanyl, tyrosyl, and phenylalanyl groups. The rotational strengths of the major near ultraviolet circular dichroic bands were significantly higher in the toxin than in low molecular weight peptides containing aromatic residues. This indicates that tetanus toxin has a stable tertiary structure.Andrew W. Mellon Foundation Teacher-Scholar Awardee.Camille and Henry Dreyfus Foundation Teacher-Scholar Awardee.     

Strongly exciton-coupled BChle chromophore system in the chlorosomal antenna of intact cells of the green bacteriumChlorobium phaeovibrioides: A spectral hole burning study

Spectral hole burning studies of intact cells of the green bacteriumChlorobium phaeovibrioides have proven that the Q_y-absorption system of antenna bacteriochlorophylle (BChle) should be interpreted in terms of the delocalized exciton level structure of an aggregate. For the first time the 0-0 band of the lowest exciton state of BChle aggregates has been directly detected as the lowest energy inhomogeneously broadened band (FWHM 100 cm^–1; position of maximum, at 739 nm) of the near-infrared BChle band in the 1.8 K excitation spectrum (FWHM=750 cm^–1; position of maximum, at 715 nm). The comparative analysis of the hole spectra, measured for the three species of BChlc- ande-containing green bacteria, has shown that the 0-0 transition bands of the lowest exciton state of BChlc ande aggregates display fundamentally similar spectral features: (1) the magnitude of inhomogeneous broadening of these bands is about 100 cm^–1; (2) at the wavelength of the maximum of each band, the amplitude of the preburnt excitation spectrum makes up 20% of the maximum amplitude of the spectrum; (3) the spectral position of each band coincides with the spectral position of the longest wavelength band of the circular dichroism spectrum; (4) the width of these bands is 2.3-times less than that of monomeric BChl in vitro.     

The Raman spectra of Bence-Jones proteins. Disulfide stretching frequencies and dependence of Raman intensity of tryptophan residues on their environments

The Raman spectra of Bence-Jones proteins (BJP) were measured for their native and denatured states. All of the native BJPs investigated gave amide I at 1670–1675 cm^?1 and amide III at 1242–1246 cm^?1. Although the amide I was shifted to 1667 cm^?1 upon the LiBr, acid, and thermal denaturation, as expected, the amide III frequency was unaltered, indicating that the antiparallel β- and disordered structures of BJP provide amide III at almost the same frequencies. The intensity of the 880-cm^?1 line of native BJP was relatively intense compared with that of amino acid mixed solution in which the mole ratios of Trp, Phe, and Tyr were adjusted to reproduce the corresponding ratios of BJP. However, the intensity was evidently reduced upon LiBr, acid, and thermal denaturation, approaching that of the amino acid mixture. Thus, the intensity of the 880-cm^?1 line is proposed as a practical probe for the environment of Trp residues. The pH dependence of the intensity of the 880-cm^?1 line suggests that one of two buried Trp residues is exposed between pH 4 and 3.2 and the other between pH 3.2 and 1.4. The variable fragment (V_L) of BJP (Tod) exhibited a S? S stretching Raman line at 525 cm^?1. Provided that the crystallographic data of the V_L of BJP is applicable to V_L of BJP (Tod), the 525 cm^?1 of the S? S stretching frequency should be assigned to a TGG conformation of linkage, but not to the AGT or AGG conformation. This supports Sugeta's model rather than Scheraga's model.     

Effect of Mid-infrared Free-Electron Laser Irradiation on Refolding of Amyloid-Like Fibrils of Lysozyme into Native Form

Aggregation of lysozyme in an acidic solution generates inactive amyloid-like fibrils, with a broad infrared peak appearing at 1,610?C1,630?cm^?1, characteristic of a ??-sheet rich structure. We report here that spontaneous refolding of these fibrils in water could be promoted by mid-infrared free-electron laser (mid-IR FEL) irradiation targeting the amide bands. The Fourier transform infrared spectrum of the fibrils reflected a ??-sheet content that was as low as that of the native structure, following FEL irradiation at 1,620?cm^?1 (amide I band); both transmission-electron microscopy imaging and Congo Red assay results also demonstrated a reduced fibril structure, and the enzymatic activity of lysozyme fibrils recovered to 70?C90?% of the native form. Both irradiations at 1,535?cm^?1(amide II band) and 1,240?cm^?1 (amide III band) were also more effective for the refolding of the fibrils than mere heating in the absence of FEL. On the contrary, either irradiation at 1,100 or 2,000?cm^?1 afforded only about 60?% recovery of lysozyme activity. These results indicate that the specific FEL irradiation tuned to amide bands is efficient in refolding of lysozyme fibrils into native form.     

Ultraviolet circular dichroism of polyproline and oriented collagen

The ultraviolet absorption, linear dichroism, circular dichroism, and oriented circular dichroism of collagen are reported and the spectra are resolved into a self-consistent set of bands in accord with exciton theory. The parallel band at 200 nm has 40% of the π → π* intensity; the perpendicular band is placed at 189 nm yielding a splitting of 2700 cm^?1. The circular dichroism is resolved into two Gaussians at λ_∥ and λ_τ (rotational strengths +14 × 10^?40 and ?32 × 10^?40 esu². cm²) plus a large non-Gaussian (“helix”) band with ampplitude ?25,000° at 201 nm. These data appear to be in reasonably good accord with recent calculations. Measurements of the absorption, linear dichroism and circular dichroism of polyproline I and II are also reported and are resolved into their component bands. Polyproline I is in good accord with exciton theory, whereas polyproline II remains unsatisfactory.     

Laser Raman spectroscopic analysis of angiotensin peptides' conformation

In this study we examined the conformation and side chain environments of angiotensins I, II, III, and [Sar¹-Ile⁵-Ala⁸]angiotensin II using laser Raman spectroscopy. The positions of the amide I bands for all four peptides were found between 1664 and 1673 cm^?1. D₂O exchange studies confirmed the positions of the amide I and amide III bands. The positions of the amide I bands for all the angiotensins were found at approximately 1665 cm^?1 and the amide III bands were all located between 1265 and 1278 cm^?1. From the positions and intensities of the amide I and III bands we concluded that all peptides share the same overall conformation consisting of β-turn structure. Spectral analysis indicated that although the spectra for all the peptides were qualitatively identical there was evidence that the angiotensin conformations were more flexible in the aqueous phase than the solid phase. Examination of the $\text{850}\text{830}$ cm^?1 tyrosine doublet suggested that the tyrosine residue in the peptides is exposed to the solvent environment and becomes more exposed as the peptide length is decreased. Therefore, there are some localized conformational differences among the angiotensins. The conformational data yielded by this study leads us to conclude that the various biological properties ascribed to the angiotensins are not due to different conformations of the peptides. The biological differences could perhaps be attributed to localized interactions of the individual amino acid residues with themselves and with the hormone receptors.     

The GABAA receptor family in the mammalian brain

The GABA_A-benzodiazepine receptor protein from bovine brain was purified by affinity chromatography and the subunit composition examined by gel electrophoresis in sodium dodecyl sulfate. Protein staining revealed a doublet at 51–53 kDa, a band at 55 kDa, and a broad band at 57–59 kDa. The 51 and 53 kDa bands co-migrated with the ₁ and ₂ gene products identified by Western blotting with subtype-specific antibodies. These two bands were also photoaffinity labeled by [³H]flunitrazepam, as was a breakdown product at 44 kDa. Partial sequencing of proteolytic fragments of these polypeptides yielded sequences found in all clones, and identified the benzodiazepine binding site within residues 8–297 and probably between 106–297 of ₁; the 44 kDa and 31 kDa bands yielded fragments containing ₃ sequence. The native ₃ polypeptide was identified with subtype-specific antibody at 57 kDa overlapping with the two major bands photolabeled with [³H]muscimol at 55 and 58 kDa. Antisera to a -selective peptide recognized four bands at 60, 58, 57 and 55 kDa. Thus, one can identify 6–8 distinct polypeptides with the possibility of another 4–6 in purified GABA_A receptor proteins, depending on brain region, consistent with the family of gene products suggested by molecular cloning.Special issues dedicated to Dr. Eugene Roberts     

Structural features and bioactivities of the chitosan

Fourier transform infrared (FT-IR) spectroscopic studies (3500-600 cm⁻¹) showed some different bands of chitosan. The absorption at 3439 cm⁻¹ is stretching vibration of -OH and -NH₂ bonds, indicating the association of the hydrogen-bond between them. The bands at 1659, 1599 and 1321 cm⁻¹ are attributable to the peaks of stretching vibrations of amide I (ν_(CO)), II (δ_(N-H)), and the peak of stretching and bending vibrations of III (ν_(C-N)) (δ_(N-H)). The chitosan showed strong free radical scavenging activities. Pretreatment with chitosan significantly prevented the decrease of antioxidant enzymes activities and the increase of p-JNK at 3 h after renal ischemia and reduced renal tubular epithelial cell apoptosis.     

Secondary Structural Changes in Globular Protein Induced by a Surfactant: Fourier Self-Deconvolution of FT-IR Spectra

Abstract

Conformational changes in ovalbumin, a globular protein, induced by an anionic surfactant, sodium dodecyl sulfate (SDS), have been monitored by an FT-IR spectrometer using ZnSe cylindrical internal reflection optics which allows high quality IR spectra to be obtained in water solution. The most notable change, on addition of SDS, occurs in the composite band of the Amide I absorption band and the vibrational frequency of the composite C=O bond shifts from 1639 cm^?1 to 1652 cm^?1. On the other hand, the position of the Amide II band remains fairly unchanged.

Comparison of the various peak positions in the deconvoluted spectra for the native protein and the perturbed protein clearly shows the effect of SDS on the secondary structures of the protein. SDS unfolds the protein. It increases the helix content slightly. More importantly, it alters the β sheet structure, destroying it almost completely in the Amide I region, while retaining it in its neighbourhood. In the deconvoluted spectra of the perturbed protein, a band at 1531 cm^?1 indicates generation of some β turns. We used the second derivative of the deconvoluted spectra for fixing positions of minor peaks and shoulders.

The results of this study indicate that the deconvolution of the normal IR spectra, consisting of composite bands, provides evidence for the specific secondary structures in a protein and for the way they are affected by changes in the environment, e.g., the addition of SDS. This makes it possible to relate conformational changes to specific secondary structures.     

Direct evidence of structural changes in reaction centers of Rb. sphaeroides containing suppressor mutations for Asp L213 → Asn: A FTIR study of QB photoreduction

In bacterial reaction centers (RCs), changes of protonation state of carboxylic groups, of quinone-protein interactions as well as backbone rearrangements occuring upon Q_B photoreduction can be revealed by FTIR difference spectroscopy. The influence of compensatory mutations to the detrimental Asp L213 Asn replacement on Q_B ^–/Q_B FTIR spectra of Rb. sphaeroides RCs was studied in three double mutants carrying a Asn M44 Asp, Arg M233 Cys, or Arg H177 His suppressor mutation. The proton uptake by Glu L212 upon Q_B ^– formation, as reflected by the positive band at 1728 cm^–1, is increased in the Asn M44 Asp and Arg H177 His suppressor RCs with respect to native RCs, and remains comparable to that observed in Asp L213 Asn mutant RCs. Only the Arg M233 Cys suppressor mutation affected the 1728 cm^–1 band, reducing its amplitude to near native level. Thus, there is no clear correlation between the apparent extent of proton uptake by Glu L212 and the recovery of the proton transfer RC function. In all of the mutant spectra, several protein (amide I and amide II) and quinone anion (C...O/C...C) modes are perturbed compared to the spectrum of native RCs. These IR data show that all of the compensatory mutations alter the semiquinone-protein interactions and the backbone providing direct evidence of structural changes accompanying the restoration of efficient proton transfer in RCs containing the Asp L213 Asn lesion.     

Investigation of adenylate kinase from Escherichia coli and its interaction with nucleotides by Fourier transform infrared spectroscopy

The secondary structure of adenylate kinase (EC 2.7.4.3) from E. coli was investigated under various conditions using Fourier transform infrared spectroscopy. The overall band contour of the conformation-sensitive amide I mode indicates that in HEPES buffer (pH 7.4) the major structure of the protein is alpha-helical. A more detailed estimate obtained from decomposition of the amide I band into its constituent component bands gives 50% alpha-helix, 26% beta-structure, 15% turns and loops, and about 9% nonrepetitive unordered structures. Binding of nucleotide (e.g., ATP) to the donor site decreases the beta-content and shifts the amide I band to higher wavenumbers, whereas binding of nucleotide (e.g., AMP) to the acceptor site does not produce any change in conformation of the protein. These results agree with the protection by ATP and lack of protection by AMP when adenylate kinase is digested with trypsin. The effect of protein denaturing agents and conditions (temperature, high pH, sodium dodecyl sulfate) on changes in the protein conformation as revealed by the conformation-sensitive amide I bands is discussed.     

Secondary structure of streptokinase in aqueous solution: a Fourier transform infrared spectroscopic study.

The secondary structure of streptokinase (Sk) in aqueous solution was quantitatively examined by using Fourier transform infrared (FT-IR) spectroscopy. Resolution enhancement techniques, including Fourier deconvolution and derivative spectroscopy, were combined with band curve-fitting procedures to quantitate the spectral information from the amide I bands. Nine component bands were found under the broad, nearly featureless amide I bands which reflect the presence of various substructures. The relative areas of these component bands indicate an amount of beta-sheet between 30 and 37% and an alpha-helix content of only 12-13% in Sk. Further conformational substructures are assigned to turns (25-26%) and to "random" structures (15-16%). Additionally, the correlation of a pronounced component band near 1640 cm-1 (10-16% fractional area) with the possible presence of 3(10)-helices is discussed.     

Second-derivative Fourier transform infrared spectra of seaweed galactans

The Fourier transform infrared spectra of agar, agarose, -, -, and -carrageenan, and ofChondrus canaliculatus, Iridaea ciliata, I. membranacea, I. laminarioides andGracilaria chilensis polysaccharides were recorded in the 4000–400 cm^-1 region. The bands in the second derivative mode are sharper and more bands are resolved than in the normal spectra.Agar, agarose andG. chilensis phycocolloids exhibit diagnostic bands at 790 and 713 cm^-1. -, - and -carrageenans, and native carrageenan-type polysaccharides fromC. canaliculatus andIridaea species exhibit bands at around 1160, 1140, 1100, 1070, 1040, 1008, 610, and 580 cm^-1. Therefore, FT-IR spectroscopy in the second-derivative mode may be applied to differentiate between agar- and carrageenan-types seaweed galactans.     

Determination of beta-turn conformation by laser Raman spectroscopy.

To correlate the Raman frequencies of the amide I and III bands to beta-turn structures, three peptides shown to contain beta-turn structure by x-ray diffraction and NMR were examined. The compounds examined were tertiary (formula: see text). The amide I band of these compounds is seen at 1,668, 1,665, and 1,677 cm-1, and the amide III band appears at 1,267, 1,265, and 1,286 cm-1, respectively. Thus, it is concluded that the amide I band for type III beta-turn structure appears in the range between 1,665 and 1,677 cm-1 and the amide III band between 1,265 and 1,286 cm-1.     

Characterization of NO adducts of the diiron center in protein R2 of<Emphasis Type="Italic"> Escherichia coli</Emphasis> ribonucleotide reductase and site-directed variants; implications for the O<Subscript>2 </Subscript>activation mechanism*

The R2 subunit of Escherichia coli ribonucleotide reductase contains a diiron site that reacts with O₂ to produce a tyrosine radical (Y122·). In wild-type R2 (R2-wt), the first observable reaction intermediate is a high-valent [Fe^III-Fe^IV] state called compound X, but in related diiron proteins such as methane monooxygenase, ⁹-desaturase, and ferritin, peroxodiiron(III) complexes have been characterized. Substitution of iron ligand D84 by E within the active site of R2 allows an intermediate (-1,2-peroxo)diiron species to accumulate. To investigate the possible involvement of a bridging peroxo species within the O₂ activation sequence of R2-wt, we have characterized the iron-nitrosyl species that form at the diiron sites in R2-wt, R2-D84E, and R2-W48F/D84E by using vibrational spectroscopy. Previous work has shown that the diiron center in R2-wt binds one NO per iron to form an antiferromagnetically coupled [{FeNO}⁷]₂ center. In the wt and variant proteins, we also observe that both irons bind one NO to form a {FeNO}⁷ dimer where both Fe–N–O units share a common vibrational signature. In the wt protein, (Fe–NO), (Fe–N–O), and (N–O) bands are observed at 445, 434 and 1742 cm^–1, respectively, while in the variant proteins the (Fe–NO) and (Fe–N–O) bands are observed ~10 cm^–1 higher and the (N–O) ~10 cm^–1 lower at 1735 cm^–1. These results demonstrate that all three proteins accommodate fully symmetric [{FeNO}⁷]₂ species with two identical Fe–N–O units. The formation of equivalent NO adducts in the wt and variant proteins strongly favors the formation of a symmetric bridging peroxo intermediate during the O₂ activation process in R2-wt.