Ligand Loop Effects on the Free Energy Change of Redox and pH-Dependent Equilibria in Cupredoxins Probed on Amicyanin Variants

Battistuzzi, Gianantonio; Borsari, Marco; Canters, Gerard W.; Rocco, Giulia Di; Waal, Ellen de; Arendsen, Yvonne; Leonardi, Alan; Ranieri, Antonio; Sola, Marcó

doi:10.1021/bi050261r

Cited by 24 publications

(45 citation statements)

References 64 publications

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“…Similar behavior has been observed in plastocyanin (25,26), pseudoazurin (27,28), and AMI (21,22,29) and is thought to have physiological relevance (26,29). Loop mutation experiments have shown that, in most cases, lengthening the ligand-containing loop lowers the His ligand pK a (9,10,12,38), whereas loop contraction increases the value (8,11,12). In AZ, the His-117 ligand does not protonate in the accessible pH range, and a pK a of Ͻ2 has been calculated (20).…”

Section: Discussionsupporting

confidence: 60%

“…Further shortening of the loop leads to an additional increase in the pK a . The structures of AZAMI and AZAMI-F demonstrate that their active-site structures, including hydrogen-bonding patterns, are very similar, and, thus, His ligand protonation must be influenced by the length of the loop [probably an entropic effect (38,39)]. The increase in the pK a of the His ligand upon shortening this loop results in a value closer to physiological pH and is, thus, more likely to have a functional role.…”

Section: Discussionmentioning

confidence: 99%

“…In AZ, the His-117 ligand does not protonate in the accessible pH range, and a pK a of Ͻ2 has been calculated (20). In the Cu(I) AZAMI loop-contraction variant, the pK a of His-115 is 5.5 [this pK a is lower than the value of 6.4 for His-96 in Cu(I) AMI] (21,38). Further shortening of the loop leads to an additional increase in the pK a .…”

Section: Discussionmentioning

confidence: 99%

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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.

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: Discussionsupporting

confidence: 60%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

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.

134

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“…Since reduction thermodynamics for metalloproteins in aqueous solution are deeply affected by solvent reorganization phenomena [21][22][23][24][25][44][45][46], the thermodynamic data listed in Table 1, as such, cannot be used to extract information on mutation-induced proteinbased effects on the thermodynamics of Fe(III) to Fe(II) reduction. In particular, it is worthy of note that for all the present KatG variants the mutation-induced changes in ΔH°′ rc and ΔS°′ rc are much larger than the corresponding changes in ΔG°′ rc , and therefore in E°′ (Table 1).…”

Section: Ferric To Ferrous Transition Of Wild-type Synechocystis Katgmentioning

confidence: 99%

“…In particular, it is worthy of note that for all the present KatG variants the mutation-induced changes in ΔH°′ rc and ΔS°′ rc are much larger than the corresponding changes in ΔG°′ rc , and therefore in E°′ (Table 1). This is typical of the presence of enthalpy-entropy compensation phenomena occurring within a series of homologous species subjected to the same reaction at fixed temperature [21,22,[42][43][44][45][46][47][48], according to Eq. (1) [45][46][47][48]:…”

Section: Ferric To Ferrous Transition Of Wild-type Synechocystis Katgmentioning

confidence: 99%

Disruption of the H-bond network in the main access channel of catalase–peroxidase modulates enthalpy and entropy of Fe(III) reduction

Vlasits

Bellei

Jakopitsch

et al. 2010

Journal of Inorganic Biochemistry

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The Importance of the Long Type 1 Copper‐Binding Loop of Nitrite Reductase for Structure and Function

Sato

Firbank

et al. 2008

Chemistry A European J

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The long 15-residue type 1 copper-binding loop of nitrite reductase has been replaced with that from the cupredoxin amicyanin (7 residues). This sizable loop contraction does not have a significant effect on the spectroscopy, and therefore, the structures of both the type 1 and type 2 Cu(II) sites. The crystal structure of this variant with Zn(II) at both the type 1 and type 2 sites has been determined. The coordination geometry of the type 2 site is almost identical to that found in the wild-type protein. However, the structure of the type 1 centre changes significantly upon metal substitution, which is an unusual feature for this class of site. The positions of most of the coordinating residues are altered of which the largest difference was observed for the coordinating His residue in the centre of the mutated loop. This ligand moves away from the active site, which results in a more open metal centre with a coordinating water molecule. Flexibility has been introduced into this region of the protein. The 200 mV increase in the reduction potential of the type 1 copper site indicates that structural changes upon reduction must stabilise the cuprous form. The resulting unfavourable driving force for electron transfer between the two copper sites, and an increased reorganisation energy for the type 1 centre, contribute to the loop variant having very little nitrite reductase activity. The extended type 1 copper-binding loop of this enzyme makes a number of interactions that are important for maintaining quaternary structure.

show abstract

Ligand Loop Effects on the Free Energy Change of Redox and pH-Dependent Equilibria in Cupredoxins Probed on Amicyanin Variants

Cited by 24 publications

References 64 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

Disruption of the H-bond network in the main access channel of catalase–peroxidase modulates enthalpy and entropy of Fe(III) reduction

The Importance of the Long Type 1 Copper‐Binding Loop of Nitrite Reductase for Structure and Function

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