Reduced forms of the iron-containing small subunit of ribonucleotide reductase from Escherichia coli

The B2 subunit of ribonucleotide reductase from Escherichia coli contains a stable tyrosyl free radical and an antiferromagnetically coupled dimeric iron center with high-spin ferric ions. The tyrosyl radical is an oxidized form of tyrosine-122. This study shows that the B2 protein has a fully reduced state, denoted reduced B2, characterized by a normal nonradical tyrosine-122 residue and a dimeric ferrous iron center. Reduced B2 can be formed either from active B2 by a three-electron reduction in the presence of suitable mediators or from apoB2 by addition of two equimolar amounts of ferrous ions in the absence of oxygen. The oxidized tyrosyl radical and the ferric iron center can be generated from reduced B2 by the admission of air. The tyrosyl radical can be selectively reduced by one-electron reduction in the presence of a suitable mediator, yielding metB2, a form that seems identical with the form resulting from treatment of active B2 with hydroxyurea. 1H NMR was used to characterize the paramagnetically shifted resonances associated with the reduced iron center. Prominent resonances were observed around 45 ppm (nonexchangeable with solvent) and 57 ppm (exchangeable with solvent) at 37 degrees C. From the temperature dependence of the chemical shifts of these resonances it was concluded that the ferrous ions in reduced B2 are only weakly, if at all, antiferromagnetically coupled. By comparison with data on the similar iron center of deoxyhemerythrin it is suggested that the 57 ppm resonance should be assigned to protons in histidine ligands of the iron center.     

Hydrogen-1 nuclear magnetic resonance of the nitrogenase iron protein (Cp2) from Clostridium pasteurianum

Proton NMR spectra (250 MHz) of the nitrogenase iron protein from Clostridium pasteurianum (Cp2) were found to display 9 or 10 paramagnetically shifted resonances in the 15-50 ppm range. The most shifted resonances belonged to two approximately equal subsets having temperature dependences of opposite sign. The latter occurrence is consistent with the interaction of the corresponding protons with an antiferromagnetically coupled metal center. The number of proton resonances of Cp2, their positions, and their temperature dependences were similar to those observed in spectra of (4Fe-4S)+ ferredoxins, particularly those of the latter that contain a single tetranuclear cluster, such as the ferredoxin from Bacillus stearothermophilus. The effects of several adenine nucleotides on the paramagnetically shifted proton resonances of Cp2 have been investigated. Whereas MgAMP had no effect at all, MgADP and MgATP were found to induce different modifications, which in both cases involved approximately half only of the shifted proton resonances. These data suggest that nucleotide binding affects mainly one part of the iron-sulfur cluster. A remarkable feature of the spectra of Cp2 in the presence of MgATP is the grouping of the shifted proton resonances in sets of two or four having identical chemical shifts and temperature dependences. A nearly perfect 2-fold symmetry is thus suggested for the arrangement of the cysteine protons around the active site. These observations lend support to the proposal that the (4Fe-4S) cluster is held symmetrically between the two identical subunits and are consistent with the existence of two MgATP binding sites on nitrogenase iron proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

1H NMR studies of porcine uteroferrin. Magnetic interactions and active site structure

Pink (reduced) uteroferrin exhibits well resolved paramagnetic NMR spectra with resonances ranging from 90 ppm downfield to 70 ppm upfield. The intensities of these signals depend on the degree of reduction and correlate well with the intensity of the EPR signals with gave = 1.74. Analyses of chemical shifts and the temperature dependence of the paramagnetically shifted resonances indicate that the Fe(III)-Fe(II) cluster in the reduced protein exhibits weak antiferromagnetic exchange coupling (-J approximately equal to 10 cm-1), in agreement with the estimate derived from the temperature dependence of the EPR signal intensity. Purple (oxidized) uteroferrin, on the other hand, exhibits no discernible paramagnetically shifted resonances, reflecting either strong antiferromagnetic coupling or an unfavorable electron spin-lattice relaxation time. Evans susceptibility comparisons between pink and purple uteroferrin show that the Fe(III)-Fe(III) cluster in the oxidized protein is more strongly coupled (-J greater than 40 cm-1). This value concurs with low temperature magnetic susceptibility measurements on both the porcine and splenic purple acid phosphatases. The isotropically shifted protons of tyrosine coordinated to the cluster are assigned by comparison with synthetic complexes. Tyrosine, earlier implicated as a ligand by resonance Raman spectroscopy, appears to coordinate only to the ferric site in pink uteroferrin. This is consistent with the relatively invariant extinction coefficients of uteroferrin in its oxidized and reduced forms and the ease of reduction of the nonchromophoric iron compared to its chromophoric partner. Other possible ligands to the cluster include histidine, suggested by the presence of downfield-shifted solvent-exchangeable resonances with appropriate isotropic shifts.     

1H NMR studies of a two-iron: two-sulfur ferredoxin from halobacterium of the dead sea

Ferredoxin isolated from Halobacterium of the Dead Sea (HFd) was found to be stable and retain its conformation in 4–0.5 M salt solutions. Reconstitution of the denatured protein to the oxidized form in ²H₂O indicated that the resonances shifted to the 8–10 ppm region, which include 18 protons, are nonexchangeable -NH protons. The C₂H and C₄H resonances of His-119 were assigned in both oxidized and reduced HFd. pH titration curves of these resonances yielded a pK_a for this His of 6.57 ± 0.1 and 6.65 ± 0.1 in oxidized and reduced HFd, respectively. pH titration curves, T₁ relaxation times, and the temperature dependence of the chemical shift were obtained for resonances between 6 and 10 ppm of oxidized HFd. In oxidized HFd a paramagnetically shifted resonance was observed at 15 ppm with 1 H intensity, and an anti-Curie temperature dependence. In reduced HFd eight resonances each with 1 H intensity were shifted downfield by 10–50 ppm and one resonance with 1 H intensity was shifted upfield to ?6.8 ppm. Four of these resonances exhibited an anti-Curie temperature dependence, two exhibited a moderate Curie dependence, and three were temperature independent.     

Purification and characterization of recombinant mouse and herpes simplex virus ribonucleotide reductase R2 subunit.

Overexpression of recombinant mouse and herpes simplex virus ribonucleotide reductase small subunit (protein R2) has been obtained by using the T7 RNA polymerase expression system. Both proteins, which constitute about 30% of the soluble Escherichia coli proteins, have been purified to homogeneity by a rapid and simple procedure. At this stage, few of the molecules contain the iron-tyrosyl free-radical center necessary for activity; however, addition of ferrous iron and oxygen under controlled conditions resulted in a mouse R2 protein containing 0.8 radical and 2 irons per polypeptide chain. In this reaction, one oxygen molecule was needed to generate each tyrosyl radical. Both proteins had full enzymatic activity. EPR spectroscopy showed that iron-center/radical interactions are considerably stronger in both mouse and viral proteins than in E. coli protein R2. CD spectra showed that the bacterial protein contains 70% alpha-helical structure compared to only about 50% in the mouse and viral proteins. Light absorption spectra between 310 and 600 nm indicate close similarity of the mu-oxo-bridged binuclear iron centers in all three R2 proteins. Furthermore, the paramagnetically shifted iron ligand proton NMR resonances show that the antiferromagnetic coupling and ligand arrangement in the iron center are nearly identical in all three species.     

Structural studies on a mitochondrial glyoxalase II

Glyoxalase 2 is a beta-lactamase fold-containing enzyme that appears to be involved with cellular chemical detoxification. Although the cytoplasmic isozyme has been characterized from several organisms, essentially nothing is known about the mitochondrial proteins. As a first step in understanding the structure and function of mitochondrial glyoxalase 2 enzymes, a mitochondrial isozyme (GLX2-5) from Arabidopsis thaliana was cloned, overexpressed, purified, and characterized using metal analyses, EPR and (1)H NMR spectroscopies, and x-ray crystallography. The recombinant enzyme was shown to bind 1.04 +/- 0.15 eq of iron and 1.31 +/- 0.05 eq of Zn(II) and to exhibit k(cat) and K(m) values of 129 +/- 10 s(-1) and 391 +/- 48 microm, respectively, when using S-d-lactoylglutathione as the substrate. EPR spectra revealed that recombinant GLX2-5 contains multiple metal centers, including a predominant Fe(III)Z-n(II) center and an anti-ferromagnetically coupled Fe(III)Fe(II) center. Unlike cytosolic glyoxalase 2 from A. thaliana, GLX2-5 does not appear to specifically bind manganese. (1)H NMR spectra revealed the presence of at least eight paramagnetically shifted resonances that arise from protons in close proximity to a Fe(III)Fe(II) center. Five of these resonances arose from solvent-exchangeable protons, and four of these have been assigned to NH protons on metal-bound histidines. A 1.74-A resolution crystal structure of the enzyme revealed that although GLX2-5 shares a number of structural features with human GLX2, several important differences exist. These data demonstrate that mitochondrial glyoxalase 2 can accommodate a number of different metal centers and that the predominant metal center is Fe(III)Zn(II).     

The effect of cytochrome P-450cam on the NMR relaxation rate of water protons.

Cytochrome P-450cam in the native, substrate-free state (Fe3+, S = 1/2) substantially reduces the NMR relaxation times, T1 and T2, of water protons. Temperature and frequency dependences of T1 and T2 were measured; they are consistent with a model of one or two protons exchanging between a binding site on a heme ligand and bulk water. The relevant parameters of this model have been deduced from the data. The spin relaxation time of the heme iron, tau S similar to 0.5 ns at 25 degrees C, is unusually long for a low spin ferric heme protein but is compatible with the line widths measured for paramagnetically shifted heme resonances. The proton residence time on the ligand, tau M similar to 1 microsecond at 25 degrees C, follows an Arrhenius law with activation energy EM similar to 15 kcal/mol. A scalar hyperfine interaction A/h = 2.2 MHz (3.1 MHz for one-proton exchange) of the found proton(s) with the heme iron is deduced from the difference between T1 and T2 observed in the fast exchange limit. The iron-proton distance is found to be 2.9 A (2.6 A for one-proton exchange). Variation of pH between pH 6.4 and 8.6 does not affect T1. The bearing of these results on the question of the axial heme ligand is discussed.     

Magnetic interaction between the tyrosyl free radical and the antiferromagnetically coupled iron center in ribonucleotide reductase

Ribonucleotide reductases from Escherichia coli and from mammalian cells are heterodimeric enzymes. One of the subunits, in the bacterial enzyme protein B2 and in the mammalian enzyme protein M2, contains iron and a tyrosyl free radical that both are essential for enzyme activity. The iron center in protein B2 is an antiferromagnetically coupled pair of high-spin ferric ions. This study concerns magnetic interaction between the tyrosyl radical and the iron center in the two proteins. Studies of the temperature dependence of electron paramagnetic resonance (EPR) relaxation and line shape reveal significant differences between the free radicals in proteins B2 and M2. The observed temperature-dependent enhanced EPR relaxation and line broadening of the enzyme radicals are furthermore completely different from those of a model UV-induced free radical in tyrosine. The results are discussed in terms of magnetic dipolar as well as exchange interactions between the free radical and the iron center in both proteins. The free radical and the iron center are thus close enough in space to exhibit magnetic interaction. For protein M2 the effects are more pronounced than for protein B2, indicating a stronger magnetic interaction.     

Proton NMR study of iron(II)-bleomycin: Assignment of resonances by saturation transfer experiments

The assignment of the paramagnetically shifted resonances of the Fe(II)-bleomycin complex in D₂O has been accomplished using the transfer of saturation method. A number of additional resonances arising from labile N$\text{H}$ protons which are shifted by the metal ion are observed in the ¹H spectrum of the complex in H₂O. The temperature dependence of the chemical shifts is consistent with the formation of an isolated 1:1 complex, but does not obey either the Curie Law or the Curie-Weiss Law. The magnitude of the shifts suggests that the valeric acid hydroxyl (or carbonyl) group, the α-amino group, the imidazole N^π, the carbamoyl oxygen, the pyrimidine N₁ and/or the secondary amino group may be coordinated to the iron(II).     

The downfield resonances in the 1H NMR spectra of Azotobacter vinelandii and Pseudomonas putida seven-iron ferredoxins

Pseudomonas putida and Azotobacter vinelandii ferredoxins each contain one [4Fe-4S] cluster and one [3Fe-4S] cluster. Their polypeptide chains are nearly identical, differing by only 15 residues out of a total of 106. T1 measurements and temperature dependence studies of the 1H NMR spectrum of each ferredoxin demonstrate that all six resolved downfield resonances are near an iron-sulfur center. The five most downfield resonances are shown to arise from protons on cysteinyl beta-carbons by incorporation of cysteine deuterated at the beta-carbon into cell protein. The sixth peak (10.5 ppm) is shown to be a non-cysteinyl proton. This peak resolves into two resonances of approximately equal intensity at temperatures below 15 degrees or above 25 degrees C. A nuclear Overhauser effect observed between the two downfield-most resonances of A. vinelandii ferredoxin indicates that they originate from a geminal pair of beta-cysteinyl protons. An Overhauser effect observed between the resonances at 22.3 and 15.7 ppm, in conjunction with other results, implies that the resonance at 22.3 ppm arises from a beta-proton on the 3Fe-center-bound Cys16, while the resonance at 15.7 ppm arises from Cys45 beta-proton, which is bound to the 4Fe center. The five most downfield resonances are pH-dependent. The sixth peak (10.5 ppm in P. putida ferredoxin) is pH-independent. Possible origins for the observed pH dependencies are discussed.     

Direct assignment of the cysteinyl, the slowly exchangeable, and the aromatic ring 1H nuclear magnetic resonances in clostridial-type ferredoxins.

We have directly assigned the 1H NMR corresponding to the cysteinyl protons, the slowly exchangeable protons, and the aromatic ring protons in the 1H NMR spectrum of Clostridium acidi-urici ferredoxin by isotopic labeling and 13C NMR decoupling techniques. We also show that the resonance pattern in the 8- to 20-ppm (from 2,2-dimethyl-2-sialapentanesulfonic acid) region of the 1H NMR spectra of oxidized Clostridium acidi-urici, Clostridium pasteurianum, Clostridium perfringens, and Peptococcus aerogenes ferredoxins are very similar, and we assign the resonances in this region by analogy with the spectrum of C. acidi-urici ferredoxin. The 1H NMR spectra of the beta protons of the cysteinyl residues of these ferredoxins differ, however, from the 1H NMR spectra of equivalent beta protons of the methylene carbon atoms bonded via a sulfur atom to [4Fe-4S] clusters in synthetic inorganic analogues. In the spectra of the synthetic compounds, the beta protons appear as a single resonance shifted 10 ppm from its unbonded reference position. In the spectra of oxidized clostridial ferredoxins, the cysteinyl beta protons appear as a series of at least eight resolved resonances with shifts that range from 6 to 14 ppm, relative to the free amino acid resonance position. This difference in the spectra of the protein and the synthetic compounds probably results from the fact that the equivalent beta protons of the synthetic compounds are not constrained and are free to rotate and thus assume the same average orientation with respect to the [4Fe-4S] cluster. The shift pattern in the 9- to 14-ppm region is identical in three different clostridial ferredoxins. This suggests that the molecular environments of the corresponding cysteinyl residues are identical. Significant differences in the resonance positions occur, however, in the 14- to 18-ppm region, suggesting that the physical environments of these cysteinyl residues differ. This may reflect differences in the orientation of the corresponding cysteinyl residues relative to the [4Fe-4S] clusters or differences in charge density at the cysteinyl beta protons or both. The slowly exchangeable protons were identified by comparing the 1H NMR spectra of ferredoxins reconstituted in H2O and 2H2O. The remaining resonances in the 8- to 20-ppm region were assigned to each of the 2 tyrosyl residues in C. acidi-urici ferredoxin. This was done by comparing the 1H NMR spectra of C. acidi-urici [(3',5'-2H2)Tyr]ferredoxin and C. acidi-urici [PHE2]ferredoxin with that of C. acidi-urici native ferredoxin.     

The iron center in ribonucleotide reductase from Escherichia coli

Ribonucleotide reductase from Escherichia coli consists of two nonidentical subunits, proteins B1 and B2. The active site is made up from both subunits. Protein B2 contains 2 iron atoms and a tyrosyl-free radical, which are essential for the enzymatic activity. The paramagnetic susceptibility of protein B2 has been measured over the temperature range 30-200 K. A deviation from the Curie law is observed at high temperatures, consistent with a structure of an antiferromagnetically coupled pair of high spin Fe(III) with an exchange coupling -J = 108(-20)+25 cm-1. Electronic spectra are resolved into components from the iron center and the radical. A band at 600 nm is clearly identified and shown to have contributions from both components. The electronic absorptions of the tyrosyl radical of protein B2 are closely similar to those reported for phenoxy radicals of tyrosine and tritertiary butyl phenol. Determinations by EPR of the amount of free radical suggest the possibility of more than one radical per active protein B2 molecule. Reconstitution of the active site from apoprotein B2 and Fe(II) is only observed in the presence of oxygen. With Fe(III), no reconstitution is obtained. The additional physical data on the iron center of protein B2 strengthen the analogy with oxidized forms of hemerythrin. The most likely structure is an antiferromagnetically coupled pair of high spin Fe(III), possibly with a bridging oxo-group.     

15N and 1H NMR study of histidine containing protein (HPr) from Staphylococcus carnosus at high pressure

The pressure-induced changes in 15N enriched HPr from Staphylococcus carnosus were investigated by two-dimensional (2D) heteronuclear NMR spectroscopy at pressures ranging from atmospheric pressure up to 200 MPa. The NMR experiments allowed the simultaneous observation of the backbone and side-chain amide protons and nitrogens. Most of the resonances shift downfield with increasing pressure indicating generalized pressure-induced conformational changes. The average pressure-induced shifts for amide protons and nitrogens are 0.285 ppm GPa(-1) at 278 K and 2.20 ppm GPa(-1), respectively. At 298 K the corresponding values are 0.275 and 2.41 ppm GPa(-1). Proton and nitrogen pressure coefficients show a significant but rather small correlation (0.31) if determined for all amide resonances. When restricting the analysis to amide groups in the beta-pleated sheet, the correlation between these coefficients is with 0.59 significantly higher. As already described for other proteins, the amide proton pressure coefficients are strongly correlated to the corresponding hydrogen bond distances, and thus are indicators for the pressure-induced changes of the hydrogen bond lengths. The nitrogen shift changes appear to sense other physical phenomena such as changes of the local backbone conformation as well. Interpretation of the pressure-induced shifts in terms of structural changes in the HPr protein suggests the following picture: the four-stranded beta-pleated sheet of HPr protein is the least compressible part of the structure showing only small pressure effects. The two long helices a and c show intermediary effects that could be explained by a higher compressibility and a concomitant bending of the helices. The largest pressure coefficients are found in the active center region around His15 and in the regulatory helix b which includes the phosphorylation site Ser46 for the HPr kinase. This suggests that this part of the structure occurs in a number of different structural states whose equilibrium populations are shifted by pressure. In contrast to the surrounding residues of the active center loop that show large pressure effects, Ile14 has a very small proton and nitrogen pressure coefficient. It could represent some kind of anchoring point of the active center loop that holds it in the right place in space, whereas other parts of the loop adapt themselves to changing external conditions.     

Spectroscopic studies of cobalt and nickel substituted rubredoxin and desulforedoxin.

The single iron site of rubredoxin was replaced by nickel and cobalt. The near-infrared/visible/UV spectra of these metal derivatives show ligand-field transitions and charge-transfer bands which closely resemble those of simple tetrathiolate complexes, indicating a tetrahedral arrangement of the sulfur cysteinyl ligands around the metal core. The 1H NMR spectra of the nickel and cobalt derivatives reveal extremely low-field contact shifted resonances of one proton intensity assigned to beta-CH2 and alpha-CH cysteinyl protons. Other well resolved resonances shifted out of the main protein spectral envelope are also observed and probably arise from contact plus pseudocontact shift mechanisms. Rubredoxins from different sulfate reducers were metal substituted and assignments of aliphatic protons are tentatively proposed, taking advantage of the amino acid sequence homologies. The present data is promising in terms of structural analysis of the coordination sphere of the metal core. It was also shown that replacement of the iron atom of desulforedoxin, a close analogue of rubredoxin, by cobalt and nickel was possible.     

A proton nuclear magnetic resonance investigation of proximal histidyl residues in human normal and abnormal hemoglobins. A probe for the heme pocket.

Proton nuclear magnetic resonance spectroscopy at 250 MHz has been used to investigate the conformations of proximal histidyl residues of human normal adult hemoglobin, hemoglobin Kempsey [beta 99(G1) Asp leads to Asn], hemoglobin Osler [beta 145(HC2) Tyr leads to Asp], and hemoglobin McKees Rocks [beta 145(HC2) Tyr leads to Term] around neutral pH in H2O at 27 degrees C, all in the deoxy form. Two resonances that occur between 58 and 76 ppm downfield from the water proton signal have been assigned to the hyperfine shifted proximal histidyl NH-exchangeable protons of the alpha- and beta-chains of deoxyhemoglobin. These two resonances are sensitive to the quaternary state of hemoglobin, amino acid substitutions in the alpha 1 beta 2-subunit interface and in the carboxy-terminal region of the beta-chain, and the addition of organic phosphates. The experimental results show that there are differences in the heme pockets among these four hemoglobins studied. The structural and dynamic information derived from the hyperfine shifted proximal histidyl NH-exchangeable proton resonances complement that obtained from the ferrous hyperfine shifted and exchangeable proton resonances of deoxyhemoglobin over the spectral region from 5 to 20 ppm downfield from H2O. The relationship between these findings and Perutz's stereochemical mechanism for the cooperative oxygenation of hemoglobin is discussed.     

Reduction of the Fe(III)-tyrosyl radical center of Escherichia coli ribonucleotide reductase by dithiothreitol

The active form of protein B2, the small subunit of ribonucleotide reductase from Escherichia coli, contains a binuclear ferric center and a free radical localized to tyrosine 122 of the polypeptide chain. MetB2 is an inactive form that lacks the tyrosine radical but retains the Fe(III) center. We earlier reported (Fontecave, M., Eliasson, R., and Reichard, P. (1989) J. Biol. Chem. 264, 9164-9170) that enzymes from E. coli interconvert B2 and metB2, possibly as part of a regulatory mechanism. Introduction of the tyrosyl radical into metB2 occurred in two steps: first, the Fe(III) center was reduced to Fe(II), generating "reduced B2"; next oxygen regenerated non-enzymatically both Fe(III) and the tyrosyl radical. Here we demonstrate that dithiothreitol (DTT) between pH 8 and 9.5 also slowly converts metB2 to B2 in the presence of oxygen. Also in this case the reaction occurs stepwise with reduced B2 as an intermediate. DTT reduces Fe(III) of both metB2 and B2. In the latter case this reaction is accompanied by the immediate loss of the tyrosyl radical. Our results indicate that the tyrosyl radical can exist only in the presence of an intact Fe(III) center. In reduced B2 iron is loosely bound to the protein, dissociates on standing and is readily removed by chelating agents. Binding decreases at higher pH. Loss of iron from reduced B2 explains why ferrous iron stimulates and iron chelators inhibit reactivation of metB2. We propose that the reactivation of mammalian ribonucleotide reductase by DTT (Thelander, M., Gr?slund, A., and Thelander, L. (1983) Biochem. Biophys. Res. Commun. 110, 859-865) may proceed via a mechanism similar to the one found here for E. coli protein B2.     

1H NMR spectra of vertebrate [2Fe-2S] ferredoxins. Hyperfine resonances suggest different electron delocalization patterns from plant ferredoxins

We report the observation of paramagnetically shifted (hyperfine) proton resonances from vertebrate mitochondrial [2Fe-2S] ferredoxins. The hyperfine signals of human, bovine, and chick [2Fe-2S] ferredoxins are described and compared with those of Anabaena 7120 vegetative ferredoxin, a plant-type [2Fe-2S] ferredoxin studied previously [Skjeldal, L., Westler, W. M., & Markley, J. L. (1990) Arch. Biochem. Biophys. 278, 482-485]. The hyperfine resonances of the three vertebrate ferredoxins were very similar to one another both in the oxidized state and in the reduced state, and slow (on the NMR scale) electron self-exchange was observed in partially reduced samples. For the oxidized vertebrate ferredoxins, hyperfine signals were observed downfield of the diamagnetic envelope from +13 to +50 ppm, and the general pattern of peaks and their anti-Curie temperature dependence are similar to those observed for the oxidized plant-type ferredoxins. For the reduced vertebrate ferredoxins, hyperfine signals were observed both upfield (-2 to -18 ppm) and downfield (+15 to +45 ppm), and all were found to exhibit Curie-type temperature dependence. This pattern and temperature dependence are distinctly different from those found with reduced plant-type ferredoxins which have signal centered around +120 ppm with Curie-type temperature dependence, assigned to cysteines which interact with Fe(III), and signals centered around +20 ppm with anti-Curie temperature dependence, assigned to cysteines which interact with Fe(II) [Dugad, L. B., La Mar, G. N., Banci, L., & Bertini, I. (1990) Biochemistry 29, 2263-2271].(ABSTRACT TRUNCATED AT 250 WORDS)     

Heteronuclear NMR studies of cobalamins. 31P NMR observations of cobalamins bound to a haptocorrin from chicken serum

A vitamin B12-binding protein (haptocorrin) from chicken serum has been purified to homogeneity by photodissociative affinity chromatography and characterized by gel electrophoresis and UV-visible spectrophotometry of its aquocobalamin, hydroxocobalamin, and cyanocobalamin complexes. The haptocorrin is a glycoprotein with a molecular mass of about 70 kDa and a protein moiety of about 40 kDa. 31P NMR resonances of the haptocorrin-cobalamin complexes are relatively broad singlets (with or without proton decoupling) shifted downfield by 0.7-1.0 ppm from the position of the free cobalamin resonances. From the line width data, the relaxation of the phosphorus nucleus is found to be dominated by chemical shift anisotropy with a very minor (13%) component from dipolar interaction with the two nearest neighbor protons. The rotational correlation time of the haptocorrin at 25 degrees C is estimated to be 85 ns and the activation energy for rotational correlation 3.9 +/- 0.3 kcal mol-1. The downfield shift of the 31P resonances of cobalamins upon binding to the haptocorrin cannot be due to hydrogen bonding phosphodiester moiety or displacement of the axial base by a group on the protein. Calculations also show that the downfield shift is very unlikely to be due to dipolar deshielding of the phosphorus nucleus by the ring current of an aromatic residue of the protein. It is concluded that the downfield shift of the 31P resonance must be due to sterically induced changes in phosphodiester conformation which may, or may not, involve steric compression of the axial Co-N bond.     

The proton magnetic resonance spectra of a cobalt(II) azurin

The proton magnetic resonance spectrum of a cobalt(II) derivative of $\text{Pseudomonas}\text{aeruginosa}$ azurin is reported. The temperature dependence of 26 resonances is described together with a study of the pH¹ titration behaviour over a range 4.7 to 9.3. A few resonances are observed shifted by more than 30 ppm from their diamagnetic positions. Of the remainder most extrapolate to the aliphatic region at T = ∞. Two lines are assigned to the C2 and C4 protons of a freely titrating histidine residue far from, and only slightly affected by, the Co(II) centre. A further two lines are assigned to the C2 hydrogen of protonated and deprotonated forms of a histidine residue in slow exchange with bulk aquaeous protons and closer to, but not bound to, the cobalt. The structure of the protein in the vicinity of the paramagnetic centre is found to be essentially insensitive to pH¹ over the range 4.7 to 9.3.     

5,6,7,8-Tetrahydrofolic acid. Conformation of the tetrahydropyrazine ring

It is suggested from analysis of proton spin-spin coupling constants that the tetrahydropyrazine ring of tetrahydrofolate is a roughly equal mixture of two half-chair conformations, one with the C-6 proton axial and the other with the C-6 proton equatorial. The chemical shifts and spin-spin coupling constants for the carbon-bound protons of (+/-)-L-, (-)-L-, and (-)-L-[6-2H] 5,6,7,8-tetrahydrofolate were measured at 25 degrees and at 300 MHZ. The resonances corresponding to the two C-7 protons in the deuterated compound constituted an AB quartet with JAB of 12 Hz and chemical shift difference of 92 Hz or 0.307 ppm; the C-7 protons are proposed to be a geminally coupled axial-equatorial pair whose rapid equilibration does not result in equivalence due to the adjacent chiral center at C-6. The spin-spin splitting in the C-7 resonances were 3.0 and 6.6 Hz for the low field and high field resonances, respectively, reflecting coupling to the C-6 proton. These coupling constants reflect the conformational equilibrium. The resonances assignable to C-9 protons are nearly equivalent in the 6-2H compound, but exhibit the resonances corresponding to a complex spin system in the 6-H compound.