Effects of mutating Asn-52 to isoleucine on the haem-linked properties of cytochrome <i>c</i>

Schejter, Abel; Koshy, Thomas I.; Luntz, Thomas L.; Sanishvili, Ruslan; Vig, Ida; Margoliash, E.

doi:10.1042/bj3020095

Cited by 14 publications

(12 citation statements)

References 36 publications

(41 reference statements)

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“…Mutations at this position have been reported to stabilize the fold of both oxidation states (21,22), with an enhanced preference for the oxidized protein (23). Contraintuitively, the elimination of the dipole of the Asn 52 side chain and its replacement by another residue induced a decrease in enthalpy of reduction.…”

Section: Resultsmentioning

confidence: 99%

“…However, this network as a whole has a sufficiently low dielectric character to differentially disfavor the oxidized iron. Indeed, it may be that because the structure has evolved in this direction that most mutations affecting the network lower the standard redox potential of the protein, indicating that the change favors the oxidized form of the cytochrome c (21).…”

Section: Resultsmentioning

confidence: 99%

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Protein Matrix and Dielectric Effect in Cytochromec

Blouin

Wallace

2001

Journal of Biological Chemistry

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The effect of the protein matrix on the standard potential of a buried redox center has been investigated by using a selection of mutants and chemical derivatives in Saccharomyces cerevisiae cytochrome c isoform 1. Assuming only local structural perturbation and no alteration of the iron-ligation chemistry, ⌬E m 0 can be regarded as a measure of the difference in polypeptide solvation of the heme charge, which reflects the dielectric properties of the protein. In double mutants Y67F/N52I Y67F/ N52V, where most of the hydrogen bond network in the heme crevice is eliminated, ⌬S redox compares to the wild type. This indicates that a fully consistent hydrogen bond network has a similar polarizability as an apolar matrix. We therefore argue that the variability in net dielectric susceptibility arises from conformational polarizability, a factor that is not a function of atomic properties and coordinates and is therefore hard to predict using conventional physical relationships.There is still much to learn about the factors determining the E m 0 Ј of redox-active proteins. Determinants of E m 0 Ј such as local structural effects (1) as well as the electrostatic landscape of the polypeptide (2) (including the protein's own charges (3, 4), dipolar matrix (5, 6), and surrounding solvent molecules (7)) have already been identified. However, implementation of this knowledge into models does not reliably reproduce experimental observations.The standard redox potential (E m 0 Ј) reflects the thermodynamics of the equilibrium between redox states. The energetics of rearrangement between redox states will therefore reflect the ability of a system to polarize in an electrostatic field. Assuming that the nature of the iron-ligand interaction remains unchanged, the values of ⌬E m 0 Ј of mutation can be regarded as a measure of change in the polarizability, or dielectric response, to the charge of the redox center.With virtually no large redox-dependent conformational change or ligand rearrangement (8 -12), cytochrome c is a suitable model to investigate the contribution of individual residues to the dielectric properties of the protein. Using three positively charged carboxyamidomethyl-methionine sulfonium ion (CAMMS) 1 derivatives 2 (Fig. 1A) generated from a mutational methionine scan (13), a series of buried mutations at position 52 (Fig. 1B) (14) and the point mutations Y67F, Y67F/ N52I, and Y67F/N52V (15), an attempt to rationalize ⌬E m 0 Ј mut in terms of polarizability of the protein matrix is made. EXPERIMENTAL PROCEDURESDetermination of Redox Potential-The measurement of redox potential was made using the method of mixtures (16) in 50 mM potassium phosphate (pH 7.0). The redox state of the sample protein was assayed in a range of redox buffers set by the ratio of ferro/ferricyanide in solution using an E m 0 Ј for the couple of ϩ0.43 V. Thermodynamics of Oxidoreduction-Temperature dependence of the equilibrium between the reduced and oxidized form of the cytochrome c was measured in 50 mM potassium phosphate (pH 7.0) wit...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Protein Matrix and Dielectric Effect in Cytochromec

Blouin

Wallace

2001

Journal of Biological Chemistry

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“…This is achieved by decreasing the cavity size around the heme and leads to lower reactivity (Schejter et al, 1994). N52l also stabilizes cytochrome c heme against degradation by H 2 O 2 (Villegas et al, 2000).…”

Section: Mutations In Evolved Hrpsmentioning

confidence: 99%

Functional expression and stabilization of horseradish peroxidase by directed evolution in Saccharomyces cerevisiae

Morawski

Quan

Arnold

2001

Biotech & Bioengineering

126

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Biotechnology applications of horseradish peroxidase (HRP) would benefit from access to tailor-made variants with greater specific activity, lower K(m) for peroxide, and higher thermostability. Starting with a mutant that is functionally expressed in Saccharomyces cerevisiae, we used random mutagenesis, recombination, and screening to identify HRP-C mutants that are more active and stable to incubation in hydrogen peroxide at 50 degrees C. A single mutation (N175S) in the HRP active site was found to improve thermal stability. Introducing this mutation into an HRP variant evolved for higher activity yielded HRP 13A7-N175S, whose half-life at 60 degrees C and pH 7.0 is three times that of wild-type (recombinant) HRP and a commercially available HRP preparation from Sigma (St. Louis, MO). The variant is also more stable in the presence of H(2)O(2), SDS, salts (NaCl and urea), and at different pH values. Furthermore, this variant is more active towards a variety of small organic substrates frequently used in diagnostic applications. Site-directed mutagenesis to replace each of the four methionine residues in HRP (M83, M181, M281, M284) with isoleucine revealed no mutation that significantly increased the enzyme's stability to hydrogen peroxide.

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“…Several studies using classical genetic procedures or site directed mutagenesis have shown that the hydrogen bond network and Wat166 modulate redox potential and the stability of the protein (13)(14)(15)(16)(17)(18)(19)(20). The high resolution three dimensional structures of the reduced and oxidized states of yeast iso-1-cytochromes c carrying mutations at position 52 and/or 67 have been recently reported (1)(2)(3).…”

mentioning

confidence: 99%

The Role of a Conserved Water Molecule in the Redox-dependent Thermal Stability of Iso-1-cytochrome c

Lett¹,

Berghuis

Frey

et al. 1996

Journal of Biological Chemistry

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Eukaryotic cytochromes c contain a buried water molecule (Wat166) next to the heme that is associated through a network of hydrogen bonds to three invariant residues: tyrosine 67, asparagine 52, and threonine 78. Single-site mutations to two of these residues (Y67F, N52I, N52A) and the double-site mutation (Y67F/N52I) were introduced into Saccharomyces cerevisiae iso-1-cytochrome c to disrupt the hydrogen bonding network associated with Wat166. The N52I and Y67F/N52I mutations lead to a loss of Wat166 while N52A and Y67F modifications lead to the addition of a new water molecule ( Water in and around proteins is recognized as being important to protein structure, function and stability (4 -6). Surface and bound water molecules have been identified in protein structures using crystallographic methods and NMR spectroscopy. Eukaryotic cytochromes c, the paradigms of electron transfer proteins, are ideally suited for investigating the structural and functional purposes of water-protein interactions (7). Crystallographic studies performed on the oxidized and reduced states of both tuna and yeast iso-1-cytochrome c proteins have indicated that a conserved and internally bound water molecule (Wat166), 1 along with the surrounding hydrogen bond network are central to the structural transition of cytochrome c between oxidation states (8, 9). The water molecule is adjacent to the heme and the hydrogen bonding network which is composed of conserved residues Asn 52 , Tyr 67 , and Thr 78 , which are also hydrogen bonded in ferrocytochrome c to the Met 80 sulfur which is one of the two heme iron ligands. The oxidation-reduction or redox potential that determines the direction of electron flow between electron transfer proteins is dependent upon the heme ligands and the surrounding peptide (10). In addition, the functional properties of cytochrome c are dependent on the oxidation state of the protein, and knowledge of the energetics of protein stability with respect to oxidation state is central to our understanding of function (11). For example, the addition of an electron to ferricytochrome c results in modified functional properties including a significant increase in stability (12).Several studies using classical genetic procedures or site directed mutagenesis have shown that the hydrogen bond network and Wat166 modulate redox potential and the stability of the protein (13-20). The high resolution three dimensional structures of the reduced and oxidized states of yeast iso-1-cytochromes c carrying mutations at position 52 and/or 67 have been recently reported (1-3). When compared to the wild type protein structure, these mutants show significant changes in their hydrogen bonding networks adjacent to the heme as well as either the displacement of the conserved internally bound Wat166 or the addition of a second internally bound water molecule.Recent investigations have shown that depending on the amino acid substitutions, varying degrees of change in the free energy of unfolding are observed for the two redox states of cyto...

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Effects of mutating Asn-52 to isoleucine on the haem-linked properties of cytochrome c

Cited by 14 publications

References 36 publications

Protein Matrix and Dielectric Effect in Cytochromec

Protein Matrix and Dielectric Effect in Cytochromec

Functional expression and stabilization of horseradish peroxidase by directed evolution in Saccharomyces cerevisiae

The Role of a Conserved Water Molecule in the Redox-dependent Thermal Stability of Iso-1-cytochrome c

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