Modulating the Redox Potential and Acid Stability of Rusticyanin by Site-Directed Mutagenesis of Ser86

Abstract

The expression of rusticyanin in Escherichia coli and a number of mutants for Ser86 is reported. Mutations of Ser86 to Asn, Asp, Gln, and Leu were undertaken as this is an Asn residue in other structurally characterized cupredoxins, and it has been suggested that this may be partly responsible for the high redox potential (680 mV) and extreme acid stability of rusticyanin. N-Terminal sequence analysis, together with other biochemical and spectrochemical characterization, shows that the recombinant wild-type protein is indistinguishable from native rusticyanin. All four mutants retain the rhombic nature of the EPR spectra and a significant absorption maximum at approximately 450 nm, thus confirming that the overall geometry of the Cu ligands is essentially maintained. The oxidized form of all four mutants is less acid stable than the wild-type protein, although the detailed mechanism of lability varies. Ser86Leu readily loses copper as the pH is reduced from 4.0, but the protein does not denature. A significant proportion (approximately 30%) of Ser86Gln is denatured at lower pH values, whereas Ser86Asn and Ser86Asp are stable as the reduced (CuI) protein. The redox potential also varies by approximately 110 mV (590-702 mV) upon these single point mutations, thus providing direct experimental support to the idea that this residue is at least in part responsible for the acid stability and the highest redox potential of rusticyanin in the cupredoxin family.

Electrostatic Effects on the Thermodynamics of Protonation of Reduced Plastocyanin

“…[32] Other important contributions come from processes of solvent reorganization that involve changes in the hydrogen-bonding network within the hydration sphere of the molecule, [17] which are mainly localized in the hydrophobic patch that surrounds the solventexposed metal-binding histidine(s), as recently indicated by molecular-dynamics and -mechanics calculations. [26] The transition thermodynamics can be split into two terms that separate the protein-based contributions (DX8' conf , where X = G, H, S) from solvent-reorganization effects (DX8' sol ): [19] …”

“…[17] For the last two species, the transition is not observed even at pH values as low as 2-3. [16,18,19] There is an open debate on the molecular factors that control the pK a values, which have been proposed to include sequence features, [5,9,[20][21][22] p-p stacking interactions, [7,9] and solvent accessibility of the metal site. [23] Gaining insight into the molecular factors that control the thermodynamics of the transition, which include formation/disruption of covalent bonds and weak interactions-the latter also involving solvent reorganization effects-is important for a deep characterization of the process.…”

NMR Hyperfine Shifts in Blue Copper Proteins: A Quantum Chemical Investigation

“…The above results indicate that all of the active site residues included in the calculations have some effects on the NMR hyperfine shifts predictions, with the axial ligands, residues hydrogen-bonded to Cys, and the carbonyl oxygen located in the fifth coordination position [35][36][37][38][39][40][41][42][43]52,53 being of particular importance. However, the results shown here also indicate that residues hydrogen-bonded to His are important and need to be taken into account to reproduce the experimental shifts.…”

Respiratory Isozyme, Two Types of Rusticyanin ofAcidithiobacillus ferrooxidans

“…It is known that the hydrophobic and electrolytic interactions in the patch surrounding the copper coordination sphere contribute to the acidstability and redox potential of this protein. 19,20,43) Theseˆve replacements may in‰uence the copper coordination center and thus account for the decrease of the molecular coe‹cient and ESR signal of the type-B protein (Fig. 4).…”

“…4,5,[10][11][12][13][14][15] This type I protein belongs to the copper protein superfamily the members of which are widely distributed in numerous organisms, 13,16) however, rusticyanin is the only member known to have both the high redox potential (＋680 mV) necessary for an electron carrier and acid stability at pH 2-3 where A. ferrooxidans thrives. 5,[17][18][19][20] The gene for rusticyanin (rus ) is well known, havingˆrst been sequenced from A. ferrooxidans strain ATCC 23270. 21) Later, the complete gene was cloned from strain ATCC 33020 22) and found to be comprised of 564 bp, encoding 155 amino acids, with additional codons encoding a 32-amino acid signal peptide.…”