Paramagnetic NMR spectroscopy and coordination structure of cobalt(II) Cys112Asp azurin

Piccioli, Mario; Luchinat, Claudio; Mizoguchi, Tadashi; Ramirez, Benjamin E.; Gray, Harry B.; Richards, John H.

doi:10.1021/ic00107a027

Cited by 64 publications

(126 citation statements)

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“…The best candidates therefore are the B-CH2 protons of the bound cysteine residue, namely Cys-87. This assignment is in agreement with the recent finding of similarly shifted signals for Co(H) azurin [22]. It is relevant to point out that in this case they are considerably less shifted (220 and 190 ppm) than the signals corresponding to Cys-112 in Co(I1) azurin (280 and 230 ppm, according to [22]).…”

Section: Discussionsupporting

confidence: 91%

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A ¹H NMR NOE study on Co(II) stellacyanin: Some clues about the structure of the metal site

Vila

1994

FEBS Letters

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The 'H NMR spectrum of Co(H) stellacyanin is reported, in which four signals not previously observed have been detected. NOE experiments were performed to assign the hyperfine shifted signals corresponding to a Cys and two His residues. Both His residues are solventaccessible and are shown to bind the metal ion through their NJ1 atoms. The j?-CH, Cys proton shifts indicate the presence of a strong axial ligand.

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Section: Discussionsupporting

confidence: 91%

“…Signals C and D are very broad, indicating that they belong to protons near to the metal center. Two candidates could be the o&o-like protons of the bound histidines, as suggested for Co(I1) azurin [22]. Signals A and B, the signals shifted the most downfield, both exhibit T, values under 0.1 ms and are dipolarly connected.…”

Section: Discussionmentioning

confidence: 92%

A ¹H NMR NOE study on Co(II) stellacyanin: Some clues about the structure of the metal site

Vila

1994

FEBS Letters

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“…7 two Leu and a Lys spin systems involving signals s-z can be drawn. Some signals of the Lys spin system are dipolarly connected with Leu86 and Gly45 signals ( Figs 7C and 4C) so, according to the azurin structure [27-351, it can be assigned to the Lys41 residue, as already reported [15]. The assignment of Leu86 and Lys41 gives additional support to the previous assignment of the Gly45 signals and allows the specific assignment of signals e and p to the a1 and a2 protons of this residue.…”

Section: Assignment Of the 1h-nmr Signalssupporting

confidence: 69%

“…ole protons of cobalt-coordinated histidines [ 3 ] . So these signals can be tentatively assigned to the CEH protons of the two coordinated histidines as already suggested [15]. Signals a-e and m-p were assigned on the basis of 2D NMR NOESY and COSY experiments to the Nt2H and C62H protons of His46 and 117 (signals a & d and b & c, respectively), the CaH, protons of Gly45 (signals e and p) and the CyH, and C w , protons of Met121 (signals f & o and m & n, respectively) [19].…”

Section: Assignment Of the 1h-nmr Signalsmentioning

confidence: 53%

“…Although this has been a common strategy in the case of the zinc proteins [l, 31, its use for blue copper proteins has been very limited up till now [3,11,15,[18][19][20]. The recent development of new assignment strategies for the NMR spectra of paramagnetic molecules as well as the improvement of the commercial NMR apparatus permit the use of the cobalt(I1) paramagnetic properties to efficiently probe the metal environment of blue copper proteins.…”

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confidence: 99%

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¹ H‐NMR Study of a Cobalt‐Substituted Blue Copper Protein: Pseudomonas Aeruginosa Co(II)‐Azurin

Salgado

Jiménez

Donaire

et al. 1995

European Journal of Biochemistry

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Substitution of copper by cobalt in blue copper proteins gives a paramagnetic metalloderivative suitable for paramagnetic NMR studies. A thorough analysis of the 'H-NMR spectrum of Pseudornonas aeruginosa Co(I1)-azurin is presented here. All the observable contact-shifted signals as well as many other paramagnetic signals from protons placed up to about 1.0 nm around the metal center, including some residues belonging to functionally important parts of the protein like the hydrophobic patch and the His35 region, have been assigned. The results obtained permit the detection and study of structural variations like those originated by the His35 ionization, and allow us to draw a feasible picture of the metal coordination site. Contact-shifted signals correspond to the same five residues which are found in the coordination sphere of the native Cu(I1)-azurin, i.e. His46, Hisll7, Cysll2, Met121 and Gly45. Among them, the histidine residues present a pattern of resonances typical for histidines coordinated to cobalt in other cobalt protein derivatives, and the cysteine signals clearly indicate a strong interaction with the paramagnetic Co(I1) ion. In contrast, the Met121 signals indicate a weak but still existent contact interaction with the metal center. On the other hand, the very weak copper ligand, Gly45, appears here as clearly coordinated to cobalt. Results are consistent with a distorted tetrahedral metal site with the cobalt deviated from the N,S plane towards the Gly45 0 axial position and weakly interacting with the Met121 sulfur.Keywords. Azurin; paramagnetic NMR; metal substitution; type 1 copper proteins ; cobalt proteins.Substitution of the naturally occumng metals in metalloproteins is used as a common strategy in inorganic biochemistry [l, 21. In a first approach it can be considered as a kind of sitedirected mutation, the most simple we can think of in the case of a metalloprotein. In this sense, apart from the possible function of the metal, it can give an idea of the metal site selectivity and structural rigidity, the mechanism of metal uptake, some general chemical properties of the metalloprotein, etc. In some special cases metal substitution is used to probe the metal site [ 1 -31. This is possible when the chromophore that brings along the exogenous metal has peculiar spectroscopic properties. For the blue copper proteins, like azurin, the natural copper(I1) center has itself singular, informative and in some way exciting spectroscopic properties (41. These are principally EPR and electronic spectral characteristics whose extensive study throughout the last few decades has achieved a detailed knowledge of type 1 copper sites [4-61. But even in this case metal substitution has proved to be useful in solving fundamental questions such as the assignment of the main visible bands of the blue copper proteins as S(Cys)-metal charge transfer bands Abbreviations. COSY, correlated spectroscopy; TOCSY, total correlation spectroscopy; NOESY, nuclear Overhauser effect spectroscopy ; WEFT, water-eliminated Fo...

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The metal site of Pseudomonas aeruginosa azurin, revealed by a crystal structure determination of the co(II) derivative and co-EPR spectroscopy

Bonander

Vänngård²,

Tsai

et al. 1997

Proteins

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The crystal structure of cobalt-substituted azurin from Pseudomonas aeruginosa has been determined to final crystallographic R value of 0.175 at 1.9 A resolution. There are four molecules in the asymmetric unit in the structure, and these four molecules are packed as a dimer of dimers. The dimer packing is very similar to that of the wild-type Pseudomonas aeruginosa azurin dimer. Replacement of the native copper by the cobalt ion has only small effects on the metal binding site presumably because of the existence of an extensive network of hydrogen bonds in its immediate neighborhood. Some differences are obvious, however. In wild-type azurin the copper atom occupies a distorted trigonal bipyramidal site, while cobalt similar to zinc and nickel occupy a distorted tetrahedral site, in which the distance to the Met121,S(delta) atom is increased to 3.3-3.5 A and the distance to the carbonyl oxygen of Gly45 has decreased to 2.1-2.4 A. The X-band EPR spectrum of the high-spin Co(II) in azurin is well resolved (apparent g values gx' = 5.23; gy' = 3.83; gz' = 1.995, and hyperfine splittings Ax' = 31; Ay' = 20-30; Az' = 53 G) and indicates that the ligand field is close to axial.

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Paramagnetic NMR spectroscopy and coordination structure of cobalt(II) Cys112Asp azurin

Cited by 64 publications

References 1 publication

A ¹H NMR NOE study on Co(II) stellacyanin: Some clues about the structure of the metal site

A ¹H NMR NOE study on Co(II) stellacyanin: Some clues about the structure of the metal site

¹ H‐NMR Study of a Cobalt‐Substituted Blue Copper Protein: Pseudomonas Aeruginosa Co(II)‐Azurin

The metal site of Pseudomonas aeruginosa azurin, revealed by a crystal structure determination of the co(II) derivative and co-EPR spectroscopy

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Paramagnetic NMR spectroscopy and coordination structure of cobalt(II) Cys112Asp azurin

Cited by 64 publications

References 1 publication

A 1H NMR NOE study on Co(II) stellacyanin: Some clues about the structure of the metal site

A 1H NMR NOE study on Co(II) stellacyanin: Some clues about the structure of the metal site

1 H‐NMR Study of a Cobalt‐Substituted Blue Copper Protein: Pseudomonas Aeruginosa Co(II)‐Azurin

The metal site of Pseudomonas aeruginosa azurin, revealed by a crystal structure determination of the co(II) derivative and co-EPR spectroscopy

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A ¹H NMR NOE study on Co(II) stellacyanin: Some clues about the structure of the metal site

A ¹H NMR NOE study on Co(II) stellacyanin: Some clues about the structure of the metal site

¹ H‐NMR Study of a Cobalt‐Substituted Blue Copper Protein: Pseudomonas Aeruginosa Co(II)‐Azurin