Active-Site Structure and Electron-Transfer Reactivity of Plastocyanins

Sato, Katsuko; Kohzuma, Takamitsu; Dennison, Christopher

doi:10.1021/ja021005u

Cited by 63 publications

(156 citation statements)

References 93 publications

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“…The protein was usually either fully reduced, fully oxidized, or contained Ϸ1:1 Cu(II):Cu(I) protein (40). The excess reductant͞oxidant was removed and the protein exchanged into 10, 20, or 100 mM phosphate, typically at pH* 8.0 (99.9% D 2 O), by using ultrafiltration (pH values quoted for deuterated solutions are uncorrected for the deuterium isotope effect and, thus, are indicated by pH*).…”

Section: Methodsmentioning

confidence: 99%

Basic requirements for a metal-binding site in a protein: The influence of loop shortening on the cupredoxin azurin

Yanagisawa

Martins

et al. 2006

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

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134

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The main active-site loop of the copper-binding protein azurin (a cupredoxin) has been shortened from C 112 TFPGH 117 SALM 121 to C 112 TPH 115 PFM 118 (the native loop from the cupredoxin amicyanin) and also to C 112 TPH 115 PM 117 . The Cu(II) site structure is almost unaffected by shortening, as is that of the Cu(I) center at alkaline pH in the variant with the C 112 TPH 115 PM 117 loop sequence. Subtle spectroscopic differences due to alterations in the spin density distribution at the Cu(II) site can be attributed mainly to changes in the hydrogen-bonding pattern. Electron transfer is almost unaffected by the introduction of the C 112 TPH 115 PFM 118 loop, but removal of the Phe residue has a sizable effect on reactivity, probably because of diminished homodimer formation. At mildly acidic pH values, the His-115 ligand protonates and dissociates from the cuprous ion, an effect that has a dramatic influence on the reactivity of cupredoxins. These studies demonstrate that the amicyanin loop adopts a conformation identical to that found in the native protein when introduced into azurin, that a shorter than naturally occurring C-terminal active-site loop can support a functional T1 copper site, that CTPHPM is the minimal loop length required for binding this ubiquitous electron transfer center, and that the length and sequence of a metal-binding loop regulates a range of structural and functional features of the active site of a metalloprotein.copper proteins ͉ electron transfer ͉ metalloproteins ͉ protein engineering N umerous approaches are being used to design metal-binding sites in proteins (1), with many of these studies informed by an understanding of the basic structural requirements for biological metal centers. Metal-binding sites in proteins are commonly formed from loops, because these regions are reasonably tolerant to sequence modifications outside of the coordinating residues (1). Cupredoxins are copper-containing electron transfer (ET) proteins that provide a significant challenge for protein-design experiments (2-4) because their scaffold is thought to constrain the metal site structure (5). In the type 1 (T1) copper sites of cupredoxins (see Fig. 1), three of the four canonical ligands Cys, His, and, usually, Met are present on a loop linking the C-terminal strands of a rigid ␤-barrel (7, 8). The fourth ligand, a His, is donated from a ␤-strand more in the core of the fold (see Fig. 1). The lengths of the metal-binding loops in known cupredoxins range from 7 to 16 residues and have a variety of primary structures. These proteins, therefore, provide a suitable system for investigating the importance of loop length and structure for the active-site integrity of a metalloprotein. Loop-directed mutagenesis has been used to swap loops between different cupredoxins, giving sites with authentic T1 properties (8)(9)(10)(11)(12). In this work, we present studies that have been aimed at assessing the structural consequences of shortening the active-site loop of a cupredoxin and have determined the short...

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Section: Methodsmentioning

confidence: 99%

Basic requirements for a metal-binding site in a protein: The influence of loop shortening on the cupredoxin azurin

Yanagisawa

Martins

et al. 2006

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

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134

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“…), was used as an example. So far, the metal site structure of this plastocyanin has not been determined, yet recent detailed spectroscopic studies (31) indicate that the structure is similar to those of other plastocyanins (9,11,22). …”

mentioning

confidence: 93%

“…In particular, the metal sites of blue copper proteins are characterized by a short coppersulfur bond. This unusual geometry is believed to be the main reason for the strong covalency of the metal site (10,16,17) and, thus, responsible for the rapid and long-range electron transfer reactivity (18)(19)(20)(21)(22) that characterizes the blue copper proteins. Detailed knowledge of the geometric and electronic metal site structures of the blue copper proteins is, therefore, imperative for understanding the function of the proteins at the molecular level.…”

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confidence: 99%

Determination of the geometric structure of the metal site in a blue copper protein by paramagnetic NMR

Hansen

Led

2006

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

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The biological function of metalloproteins is closely tied to the geometric and electronic structures of the metal sites. Here, we show that the geometric structure of the metal site of a metalloprotein in solution can be determined from experimentally measured electron-nuclear spin-spin interactions obtained by NMR. Thus, the geometric metal site structure of plastocyanin from Anabaena variabilis was determined by including the paramagnetic relaxation enhancement of protons close to the copper site as restraints in a conventional NMR structure determination, together with the distribution of the unpaired electron onto the ligand atoms. Also, the interproton distances (nuclear Overhauser enhancements) and dihedral angles (scalar nuclear spin-spin couplings) normally used in NMR structure determinations were included as restraints. The structure calculations were carried out with the program X-PLOR and a module that takes into account the specific characteristics of the paramagnetic restraints. A well defined metal site structure was obtained with the structural characteristics of the blue copper site, including a distorted tetrahedral geometry, a short Cu-Cys S ␥ bond, and a long Cu-Met S ␦ bond. Overall, the agreement of the obtained metal site structure of Anabaena variabilis plastocyanin with those of other plastocyanins obtained by x-ray crystallography confirms the reliability of the approach.metal site structure ͉ metalloproteins ͉ paramagnetic nuclear relaxation T he biological function of many metalloproteins stems from the geometric and electronic structures of the metal sites imposed by the protein environment. In the blue copper proteins, such as plastocyanins, amicyanins, and azurins, the geometry of the copper site is unusual as compared with smallmolecule copper complexes (1-15). In particular, the metal sites of blue copper proteins are characterized by a short coppersulfur bond. This unusual geometry is believed to be the main reason for the strong covalency of the metal site (10,16,17) and, thus, responsible for the rapid and long-range electron transfer reactivity (18-22) that characterizes the blue copper proteins. Detailed knowledge of the geometric and electronic metal site structures of the blue copper proteins is, therefore, imperative for understanding the function of the proteins at the molecular level.So far, the geometric structure of the metal site in blue copper proteins (12-14) has been determined primarily by x-ray crystallography and extended x-ray absorption fine structure (3,23,24), whereas the electronic structure of the blue copper site has been determined theoretically from quantum chemical calculations (5, 25-27) and experimentally by x-ray absorption spectroscopy (16, 17), and more recently by nuclear paramagnetic relaxation (28-31). These structures have formed the basis for a detailed understanding of the biological function of the proteins by elucidating the interplay between the electronic and geometric structure of the metal site (8,15,(32)(33)(34). However, the geomet...

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“…Such oxidations catalyzed by transition metal complexes are of importance in biological and industrial processes. A diverse variety of copper metalloenzymes are known, including azurins [1], laccases [2] and plastocyanins [3], which are involved in electron transfer reactions. Ascorbate oxidases [4] are used as oxidizing enzymes, while hemocyanins [5,6] are copper-containing oxygen transport proteins found in arthropods and mollusks.…”

Section: Introductionmentioning

confidence: 99%

A polymer-supported diacetatobis(2-quinolylbenzimidazole)copper(II) complex as an efficient catalyst for oxidation of alcohols with tert-butyl hydroperoxide

Shilpa

Gayathri

2012

Transition Met Chem

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A polymer-supported diacetatobis(2-quinolylbenzimidazole)copper(II) complex [PS-(QBIM) 2 Cu(II)] was synthesized by functionalization of chloromethylated polystyrene cross-linked with 6.5 % divinyl benzene with 2-(2 0 -quinolyl)benzimidazole and subsequent treatment with Cu(OAc) 2 in methanol. The complex was characterized by physical, analytical and spectroscopic techniques. Electronic and ESR spectra, together with magnetic susceptibility measurements, indicated that the complex was paramagnetic with distorted octahedral geometry around the copper. The complex was found to be active toward oxidation of various alcohols including phenol, benzyl alcohol and cyclohexanol using 70 % aqueous tert-butyl hydroperoxide under mild conditions. Under the optimized reaction conditions, cyclohexanol gave 100 % conversion to cyclohexanone, benzyl alcohol gave 98 % yield of benzaldehyde and phenol gave 89 % yield of catechol and 4 % of hydroquinone. The complex was recycled five times without much loss in catalytic activity.

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Active-Site Structure and Electron-Transfer Reactivity of Plastocyanins

Cited by 63 publications

References 93 publications

Basic requirements for a metal-binding site in a protein: The influence of loop shortening on the cupredoxin azurin

Basic requirements for a metal-binding site in a protein: The influence of loop shortening on the cupredoxin azurin

Determination of the geometric structure of the metal site in a blue copper protein by paramagnetic NMR

A polymer-supported diacetatobis(2-quinolylbenzimidazole)copper(II) complex as an efficient catalyst for oxidation of alcohols with tert-butyl hydroperoxide

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