2D NMR Approaches to Characterizing the Molecular Structure and Dynamic Stability of the Active Site for Cyanide-Inhibited Horseradish Peroxidase

Chen, Zhigang; Ropp, Jeffrey S. De; Hernández, Griselda; Mar, Gerd N. La

doi:10.1021/ja00098a040

Cited by 69 publications

(131 citation statements)

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“…EXSY and NOESY experiments were performed in a phase-sensitive mode using the WEFT-NOESY pulse sequence (27) with delays, , of 25-50 ms between the 180°and 90°pulses and with mixing times of 3-10 ms. The spectra were Fourier-transformed using 512 or 1024 data points in both dimensions and square sine-bell weighting functions shifted 60 or 80°.…”

Section: Methodsmentioning

confidence: 99%

The Dynamic Properties of the M121H Azurin Metal Site as Studied by NMR of the Paramagnetic Cu(II) and Co(II) Metalloderivatives

Salgado¹,

Kroes²,

Berg³

et al. 1998

Journal of Biological Chemistry

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The M121H azurin mutant in solution presents various species in equilibrium that can be detected and studied by 1 H NMR of the Cu(II) and Co(II) paramagnetic metalloderivatives. In both cases up to three species are observed in slow exchange, the proportions of which are different for the two metalloderivatives. Above pH 5 the major species displays a tetrahedral coordination in which the His 121 can be observed as a coordinated residue. Its metal site corresponds to a new type of site that is defined as a type 1.5 site. The second and third species resemble the wild type (type 1) azurin and, above pH 4.5, they are present only at a low concentration. At low pH a protonation process increases the proportion of both type 1 species at the expense of the type 1.5 species. This process, characterized by a pK a ‫؍‬ 4.3, is assigned to the protonation of His 121 . At high pH the NMR spectrum of the Co(II)-M121H azurin experiences an additional transition, which is not observed in the case of the Cu(II) protein. The dynamic properties of the M121H metal site appear to be related to changes in the coordination geometry and the strength of the axial interaction between the N ␦1 (His 121 ) and the metal.An important challenge in protein engineering is the creation or modification of metal sites in proteins (1, 2). Properties that one might wish to manipulate range from metal binding strength and metal site stability to redox potential, catalytic activity, and substrate specificity. One way to meet this task is the creation of novel metal binding sites in proteins or protein domains that in their native state have no such site (3-7). This approach depends heavily on computer-assisted modeling and automated searches for appropriate attachment sites of side chains that can act as metal ligands. The side chains must be able to attain an orientation that is favorable for strong metal binding while at the same time leaving sufficient room for possible substrate binding. The method is demanding and success is not within easy reach, but when successful the result can be spectacular (3-7).A second approach makes use of natural metalloproteins and modifies existing metal sites by applying changes in the first and second coordination shell of the metal (1, 8). The advantage of this approach is that it is relatively easy to implement and that the native site provides a solid background against which

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Section: Methodsmentioning

confidence: 99%

The Dynamic Properties of the M121H Azurin Metal Site as Studied by NMR of the Paramagnetic Cu(II) and Co(II) Metalloderivatives

Salgado¹,

Kroes²,

Berg³

et al. 1998

Journal of Biological Chemistry

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“…Clean total correlation spectroscopy (clean-TOCSY) [30] experiments were performed using the MLEV-17 pulse sequence with spin lock applied for 40 and 15 ms and a recycle time of 1.5 s. Nuclear Overhauser effect spectroscopy (NOESY) [31] experiments were recorded using mixing and recycle delays in the range 15-150 ms and 300 ms-1.5 s, respectively. In order to improve the intensity of the connectivities between fast-relaxing signals, a weftNOESY experiment (180-zl-90-t,-90-z,-90-AQ-RD) [32] was also recorded in H,O with values of 95, 15, and 230 ms, for the zl, z, , and the recycle time, respectively. The water signal was suppressed by presaturation during the mixing time and, in the NOESY-type experiments, also in the relaxation delay.…”

Section: Methodsmentioning

confidence: 99%

Solution Structure of the Oxidized 2[4Fe‐4S] Ferredoxin from Clostridium Pasteurianum

Bertini

Donaire

Feinberg

et al. 1995

European Journal of Biochemistry

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Following the recently developed approach to the solution structure of paramagnetic high-potential iron-sulfur proteins, the three-dimensional structure in solution of the oxidized Clostridium pasteurianum ferredoxin has been solved by 'H-NMR. The X-ray structure is not available. The protein contains 55 amino acids and two clusters. In the oxidized state, the clusters have S = 0 ground states, but are paramagnetic because of thermal population of excited states. Due to the somewhat small size of the protein and to the presence of two clusters, approximately 55% of the residues have at least one proton with a non-selective T , smaller than 25 ms.The protein has thus been used as a test system to challenge the present paramagnetic NMR methodology both in achieving an extended assignment and in obtaining a suitable number of constraints. 79% of protein protons have been assigned. Analogy with other ferredoxins of known structure has been of help to speed up the final stages of the assignment, although we have shown that this independent information is not necessary. In addition to dipolar connectivities, partially detected through tailored experiments, 3Jm.H0, H-bond constraints and dihedral angle constraints on the Cys x2 angles have been generated by using a recently derived Karplus-type relationship for the hyperfine shifts of cysteine PCH, protons. In total, 456 constraints have been used in distance geometry calculations. The final quality of the structures is satisfactory, with root-mean-square deviation values of 66 pm and 108 pm for backbone and heavy atoms, respectively. The resulting structure is compared with that of Clostridium acidi uriciBiol. 243, 683-6951. The two proteins are very similar in the overall folding, secondary structure elements and side-chain orientations. The Ca root-mean-square deviation values between the X-ray-determined C. acidi urici ferredoxin structure and the conformer with lowest energy of the C. pasteurianum ferredoxin family is 78 pm (residues 3-53). Discrepancies in residues 26-28 may arise from the disorder observed in the X-ray structure in that region.Keywords: NMR ; solution structure ; paramagnetic metalloproteins ; ferredoxins ; iron-sulfur proteins.It has been recently shown that it is possible to obtain threedimensional structures of paramagnetic proteins in solution [ 1, 21 (Bertini, I., Eltis, D., Felli, I. C., Kastrau, D. H. W., Luchinat, C. and Piccioli, M., unpublished results). This can be achieved by combining classical NMR methods for structure determination [3-51 with tailored experiments to detect connectivities involving fast relaxing signals [6-101. It is known that the feasibility of a solution structure in the case of diamagnetic systems is dependent essentially on the molecular mass of the protein [1 11. In paramagnetic proteins, the feasibility crucially depends on the percentage of nuclei affected by paramagnetism.The solution structures of two 4Fe-4S high-potential ironsulfur proteins (Hipips), in both oxidation states, have been reCorrespondence...

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“…Compared with the extensive and in depth NMR studies on CcP (28 -36) and horseradish peroxidase (34,(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53), relatively few NMR studies of CPO have been reported (54 -58). Furthermore, the most powerful NMR approach, the two-dimensional NMR technique that has led to the unambiguous assignment of major hyperfine-shifted signals in a number of heme peroxidases (59), has not been applied to the investigation of CPO.…”

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confidence: 99%

Two-dimensional NMR Study of the Heme Active Site Structure of Chloroperoxidase

Wang

Tachikawa

et al. 2003

Journal of Biological Chemistry

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2D NMR Approaches to Characterizing the Molecular Structure and Dynamic Stability of the Active Site for Cyanide-Inhibited Horseradish Peroxidase

Cited by 69 publications

References 1 publication

The Dynamic Properties of the M121H Azurin Metal Site as Studied by NMR of the Paramagnetic Cu(II) and Co(II) Metalloderivatives

The Dynamic Properties of the M121H Azurin Metal Site as Studied by NMR of the Paramagnetic Cu(II) and Co(II) Metalloderivatives

Solution Structure of the Oxidized 2[4Fe‐4S] Ferredoxin from Clostridium Pasteurianum

Two-dimensional NMR Study of the Heme Active Site Structure of Chloroperoxidase

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