Calculation of the Redox Potential of the Protein Azurin and Some Mutants

Azurin from Pseudomonas aeruginosa is a small 128‐residue, copper‐containing protein. Its redox potential can be modified by mutating the protein. Free‐energy calculations based on classical molecular‐dynamics simulations of the protein and from mutants in aqueous solution at different pH values were used to compute relative redox potentials. The precision of the free‐energy calculations with the λ coupling‐parameter approach is evaluated as function of the number and sequence of λ values, the sampling time and initial conditions. It is found that the precision is critically dependent on the relaxation of hydrogen‐bonding networks when changing the atomic‐charge distribution due to a change of redox state or pH value. The errors in the free energies range from 1 to 10 kBT, depending on the type of process. Only qualitative estimates of the change in redox potential by protein mutation can be obtained.

[1]  K. Schulten,et al.  Free energy calculation from steered molecular dynamics simulations using Jarzynski's equality , 2003 .

[2]  Alan E. Mark,et al.  Calculation of Relative Free-Energy Via Indirect Pathways , 1991 .

[3]  R. Huber,et al.  Crystal structure analysis of oxidized Pseudomonas aeruginosa azurin at pH 5.5 and pH 9.0. A pH-induced conformational transition involves a peptide bond flip. , 1991, Journal of molecular biology.

[4]  Alan E. Mark,et al.  The GROMOS96 Manual and User Guide , 1996 .

[5]  H. Nar,et al.  Structure of the azurin mutant Phe114Ala from Pseudomonas aeruginosa at 2.6 A resolution. , 1995, Acta crystallographica. Section D, Biological crystallography.

[6]  H. Berendsen,et al.  Molecular dynamics with coupling to an external bath , 1984 .

[7]  C. Jarzynski,et al.  A “fast growth” method of computing free energy differences , 2001 .

[8]  R. Huber,et al.  Complete sequential 1H and 15N nuclear magnetic resonance assignments and solution secondary structure of the blue copper protein azurin from Pseudomonas aeruginosa. , 1992, Biochemistry.

[9]  D. van der Spoel,et al.  GROMACS: A message-passing parallel molecular dynamics implementation , 1995 .

[10]  J. Kirkwood.  Statistical Mechanics of Fluid Mixtures , 1935 .

[11]  W F van Gunsteren,et al.  Decomposition of the free energy of a system in terms of specific interactions. Implications for theoretical and experimental studies. , 1994, Journal of molecular biology.

[12]  T. N. Bhat,et al.  The Protein Data Bank , 2000, Nucleic Acids Res..

[13]  Matteo Pappalardo,et al.  Free energy perturbation and molecular dynamics calculations of copper binding to azurin , 2003, J. Comput. Chem..

[14]  Edward N. Baker,et al.  Copper coordination geometry in azurin undergoes minimal change on reduction of copper(II) to copper(I) , 1990 .

[15]  Irwin Oppenheim,et al.  Statistical Mechanical Theory of Transport Processes. VII. The Coefficient of Thermal Conductivity of Monatomic Liquids , 1954 .

[16]  Ranbir Singh,et al.  J. Mol. Struct. (Theochem) , 1996 .

[17]  Arieh Warshel,et al.  Frozen density functional free energy simulations of redox proteins: computational studies of the reduction potential of plastocyanin and rusticyanin. , 2003, Journal of the American Chemical Society.

[18]  Xiaodong Zhang,et al.  CALCULATING ACCURATE REDOX POTENTIALS IN ENZYMES WITH A COMBINED QM/MM FREE ENERGY PERTURBATION APPROACH , 2002 .

[19]  A. Gewirth,et al.  Electronic structure of the oxidized and reduced blue copper sites: contributions to the electron transfer pathway, reduction potential, and geometry☆ , 1996 .

[20]  Thomas B. Woolf,et al.  Improving the efficiency and reliability of free energy perturbation calculations using overlap sampling methods , 2004, J. Comput. Chem..

[21]  Berk Hess,et al.  LINCS: A linear constraint solver for molecular simulations , 1997 .

[22]  H. Berendsen,et al.  Interaction Models for Water in Relation to Protein Hydration , 1981 .

[23]  W F van Gunsteren,et al.  Can the stability of protein mutants be predicted by free energy calculations? , 1993, Protein engineering.

[24]  H. Nar,et al.  Structure of Pseudomonas aeruginosai zinc azurin mutant Asn47Asp at 2.4 A resolution. , 1993, Acta crystallographica. Section D, Biological crystallography.

[25]  B. Rabenstein,et al.  Artificial cytochrome b: computer modeling and evaluation of redox potentials. , 2001, Journal of the American Chemical Society.

[26]  T. Woolf,et al.  Staging Is More Important than Perturbation Method for Computation of Enthalpy and Entropy Changes in Complex Systems , 2003 .

[27]  Wei Yang,et al.  Free energy simulations: use of reverse cumulative averaging to determine the equilibrated region and the time required for convergence. , 2004, The Journal of chemical physics.

[28]  R. Zwanzig.  High‐Temperature Equation of State by a Perturbation Method. I. Nonpolar Gases , 1954 .

[29]  D. Salahub,et al.  QM/MM calculations of EPR hyperfine coupling constants in blue copper proteins , 2003 .

[30]  Jan H. Jensen,et al.  Determinants of the relative reduction potentials of type-1 copper sites in proteins. , 2004, Journal of the American Chemical Society.

[31]  L. H. Jensen,et al.  Structural Features of Azurin at 2.7 Angstroms Resolution , 1980 .

[32]  Qiang Cui,et al.  Free energy perturbation calculations with combined QM/MM potentials complications, simplifications, and applications to redox potential calculations , 2003 .

[33]  M. Sola,et al.  Redox thermodynamics of blue copper proteins , 1999 .

[34]  J. Sanders-Loehr,et al.  Effect of lysine ionization on the structure and electrochemical behaviour of the Met44-->Lys mutant of the blue-copper protein azurin from Pseudomonas aeruginosa. , 1993, European journal of biochemistry.

[35]  H. Gray,et al.  Spectroelectrochemistry of blue copper proteins: pH and temperature dependences of the reduction potentials of five azurins , 1992 .

[36]  Berk Hess,et al.  GROMACS 3.0: a package for molecular simulation and trajectory analysis , 2001 .

[37]  S. Wijmenga,et al.  Backbone dynamics of azurin in solution: slow conformational change associated with deprotonation of histidine 35. , 1999, Biochemistry.

[38]  H. Berendsen,et al.  THERMODYNAMICS OF CAVITY FORMATION IN WATER - A MOLECULAR-DYNAMICS STUDY , 1982 .

[39]  Ulf Ryde,et al.  Combined quantum and molecular mechanics calculations on metalloproteins. , 2003, Current opinion in chemical biology.

[40]  S. Cannistraro,et al.  Concerted motions in copper plastocyanin and azurin: an essential dynamics study. , 2001, Biophysical chemistry.

[41]  Wilfred F. van Gunsteren,et al.  Computer Simulation of Biomolecular Systems: Theoretical and Experimental Applications , 1989 .

[42]  F. Matthias Bickelhaupt,et al.  Chemistry with ADF , 2001, J. Comput. Chem..

[43]  David A. Kofke,et al.  Accuracy of free-energy perturbation calculations in molecular simulation. I. Modeling , 2001 .

[44]  M Mezei,et al.  Free Energy Simulations a , 1986, Annals of the New York Academy of Sciences.

[45]  Lukas D. Schuler,et al.  On the Choice of Dihedral Angle Potential Energy Functions for n-Alkanes , 2000 .

[46]  R. Guzzi,et al.  Active site modeling in copper azurin molecular dynamics simulations , 2004, Journal of molecular modeling.

[47]  E. T. Adman,et al.  Structural Features of Azurin at 2.7 Å Resolution , 1981 .

[48]  X. Daura,et al.  Parametrization of aliphatic CHn united atoms of GROMOS96 force field , 1998 .

[49]  David A. Kofke,et al.  Accuracy of free-energy perturbation calculations in molecular simulation. II. Heuristics , 2001 .

[50]  Marcel Swart,et al.  A charge analysis derived from an atomic multipole expansion , 2001 .

[51]  M. Karplus,et al.  Electrostatic effects of charge perturbations introduced by metal oxidation in proteins. A theoretical analysis. , 1988, Journal of molecular biology.

[52]  A. Bizzarri.  TOPICAL REVIEW: Neutron scattering and molecular dynamics simulation: a conjugate approach to investigate the dynamics of electron transfer proteins , 2004 .

[53]  Wilfred F. van Gunsteren,et al.  Computation of free energy , 2002 .

[54]  F. Young.  Biochemistry , 1955, The Indian Medical Gazette.

[55]  M. C. Feiters,et al.  The pH and redox-state dependence of the copper site in azurin from Pseudomonas aeruginosa as studied by EXAFS. , 1986, Biochimica et biophysica acta.