Azurin is a small blue copper protein that participates in redox reactions during anaerobic respiration in Pseudomonas aeruginosa, and there are a significant number of studies employing this model to investigate the electron transfer (ET) processes or coordination sphere of metal ion in metalloproteins. Azurin naturally contains Cu(II/I) as a central ion and is redox-active for a single electron ET. Moreover, azurin with no central ion (apo-azurin) is capable of binding other metal cofactors-e.g., Zn(II)-forming redox-inactive Zn-form and many others impacting the redox potential and structural variation in the active site's arrangement. Also, mutations of amino acid residues in the immediate vicinity of the metal ion can influence the structure and functionality of a particular metalloprotein. Therefore, this review aims to summarize the abundant information about selected topics related to redox reactions and blue copper proteins, particularly azurin, and is structured as follows: (i) introduction to the structure, properties, and physiological role of this group of metalloproteins, (ii) the role of the equatorial and axial ligands of the central metal ions, or metal species, in the active site on the metal coordination sphere's structure and related determination of the particular azurin form's redox potentials, and (iii) the effects of the particular amino acid's moiety (Phe, Tyr and Trp residues together with acceleration employing Trp-Trp π-π stacking interactions contrary to ET distance dependence) on the preferable type of long-range ET mechanism in an azurin-mediated model biomolecule. We assume that azurin is a suitable model to study the structural functionality of a particular central metal ion or individual amino acid residues in the central ion coordination sphere for studying the redox potential and ET reactions in metalloproteins.

Department of Biochemistry Faculty of Science Charles University Hlavova 2030 CZ 12843 Prague 2 Czech Republic

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Gray H.B., Winkler J.R. Electron Transfer in Proteins. Annu. Rev. Biochem. 1996;65:537–561. doi: 10.1146/annurev.bi.65.070196.002541. PubMed DOI

Fereiro J.A., Yu X., Pecht I., Sheves M., Cuevas J.C., Cahen D. Tunneling Explains Efficient Electron Transport via Protein Junctions. Proc. Natl. Acad. Sci. USA. 2018;115:E4577–E4583. doi: 10.1073/pnas.1719867115. PubMed DOI PMC

Winkler J.R., Gray H.B. Long-Range Electron Tunneling. J. Am. Chem. Soc. 2014;136:2930–2939. doi: 10.1021/ja500215j. PubMed DOI PMC

Warren J.J., Ener M.E., Vlček A., Jr., Winkler J.R., Gray H.B. Electron Hopping through Proteins. Coord. Chem. Rev. 2012;256:2478–2487. doi: 10.1016/j.ccr.2012.03.032. PubMed DOI PMC

Hosseinzadeh P., Marshall N.M., Chacón K.N., Yu Y., Nilges M.J., New S.Y., Tashkov S.A., Blackburn N.J., Lu Y. Design of a Single Protein That Spans the Entire 2-V Range of Physiological Redox Potentials. Proc. Natl. Acad. Sci. USA. 2016;113:262–267. doi: 10.1073/pnas.1515897112. PubMed DOI PMC

Liu J., Chakraborty S., Hosseinzadeh P., Yu Y., Tian S., Petrik I., Bhagi A., Lu Y. Metalloproteins Containing Cytochrome, Iron–Sulfur, or Copper Redox Centers. Chem. Rev. 2014;114:4366–4469. doi: 10.1021/cr400479b. PubMed DOI PMC

Nersissian A.M., Shipp E.L. Blue Copper-Binding Domains. Adv. Protein Chem. 2002;60:271–340. doi: 10.1016/S0065-3233(02)60056-7. PubMed DOI

Savelieff M.G., Wilson T.D., Elias Y., Nilges M.J., Garner D.K., Lu Y. Experimental Evidence for a Link among Cupredoxins: Red, Blue, and Purple Copper Transformations in Nitrous Oxide Reductase. Proc. Natl. Acad. Sci. USA. 2008;105:7919–7924. doi: 10.1073/pnas.0711316105. PubMed DOI PMC

Harris R.L., Eady R.R., Samar Hasnain S., Gary Sawers R. Coordinate Synthesis of Azurin I and Copper Nitrite Reductase in Alcaligenes Xylosoxidans during Denitrification. Arch. Microbiol. 2006;186:241–249. doi: 10.1007/s00203-006-0139-z. PubMed DOI

Pérez-Henarejos S.A., Alcaraz L.A., Donaire A. Blue Copper Proteins: A Rigid Machine for Efficient Electron Transfer, a Flexible Device for Metal Uptake. Arch. Biochem. Biophys. 2015;584:134–148. doi: 10.1016/j.abb.2015.08.020. PubMed DOI

Colman P.M., Freeman H.C., Guss J.M., Murata M., Norris V.A., Ramshaw J.A.M., Venkatappa M.P. X-Ray Crystal Structure Analysis of Plastocyanin at 2.7 Å Resolution. Nature. 1978;272:319–324. doi: 10.1038/272319a0. DOI

Guss J.M., Bartunik H.D., Freeman H.C. Accuracy and Precision in Protein Structure Analysis: Restrained Least-Squares Refinement of the Structure of Poplar Plastocyanin at 1.33 Å Resolution. Pt 6Acta Crystallogr. B. 1992;48:790–811. doi: 10.1107/S0108768192004270. PubMed DOI

Nar H., Messerschmidt A., Huber R., van de Kamp M., Canters G.W. Crystal Structure Analysis of Oxidized Pseudomonas Aeruginosa Azurin at pH 5·5 and pH 9·0. J. Mol. Biol. 1991;221:765–772. doi: 10.1016/0022-2836(91)80173-R. PubMed DOI

Bank R.P.D. RCSB PDB—1E5Y: Azurin from Pseudomonas Aeruginosa, Reduced Form, pH 5.5. [(accessed on 3 February 2025)]. Available online: https://www.rcsb.org/structure/1e5y.

Estrada E., Uriarte E. Folding Degrees of Azurins and Pseudoazurins: Implications for Structure and Function. Comput. Biol. Chem. 2005;29:345–353. doi: 10.1016/j.compbiolchem.2005.06.005. PubMed DOI

De Rienzo F., Gabdoulline R.R., Menziani M.C., Wade R.C. Blue Copper Proteins: A Comparative Analysis of Their Molecular Interaction Properties. Protein Sci. Publ. Protein Soc. 2000;9:1439–1454. doi: 10.1110/ps.9.8.1439. PubMed DOI PMC

Tsai L.-C., Sjölin L., Langer V., Bonander N., Karlsson B.G., Vänngård T., Hammann C., Nar H. Structure of the Azurin Mutant Nickel–Trp48Met from Pseudomonas Aeruginosa at 2.2 Å Resolution. Acta Crystallogr. D Biol. Crystallogr. 1995;51:711–717. doi: 10.1107/S0907444995001041. PubMed DOI

Silvestrini M.C., Falcinelli S., Ciabatti I., Cutruzzolà F., Brunori M. Pseudomonas Aeruginosa Nitrite Reductase (or Cytochrome Oxidase): An Overview. Biochimie. 1994;76:641–654. doi: 10.1016/0300-9084(94)90141-4. PubMed DOI

van Gastel M., Coremans J.W.A., Mol J., Jeuken L.J.C., Canters G.W., Groenen E.J.J. The Binding of Imidazole in an Azurin-like Blue-Copper Site. JBIC J. Biol. Inorg. Chem. 1999;4:257–265. doi: 10.1007/s007750050311. PubMed DOI

Leckner J., Bonander N., Wittung-Stafshede P., Malmström B.G., Karlsson B.G. The Effect of the Metal Ion on the Folding Energetics of Azurin: A Comparison of the Native, Zinc and Apoprotein. Biochim. Biophys. Acta BBA-Protein Struct. Mol. Enzymol. 1997;1342:19–27. doi: 10.1016/S0167-4838(97)00074-5. PubMed DOI

Rosen P., Pecht I. Conformational Equilibria Accompanying the Electron Transfer between Cytochrome c (P551) and Azurin from Pseudomonas Aeruginosa. Biochemistry. 1976;15:775–786. doi: 10.1021/bi00649a008. PubMed DOI

Dennison C. Investigating the Structure and Function of Cupredoxins. Coord. Chem. Rev. 2005;249:3025–3054. doi: 10.1016/j.ccr.2005.04.021. DOI

Gewirth A.A., Solomon E.I. Electronic Structure of Plastocyanin: Excited State Spectral Features. J. Am. Chem. Soc. 1988;110:3811–3819. doi: 10.1021/ja00220a015. DOI

van de Kamp M., Hali F.C., Rosato N., Agro A.F., Canters G.W. Purification and Characterization of a Non-Reconstitutable Azurin, Obtained by Heterologous Expression of the Pseudomonas Aeruginosa Azu Gene in Escherichia Coli. Biochim. Biophys. Acta. 1990;1019:283–292. doi: 10.1016/0005-2728(90)90206-J. PubMed DOI

Margoliash E., Schejter A. Cytochrome c. In: Anfinsen C.B., Anson M.L., Edsall J.T., Richards F.M., editors. Advances in Protein Chemistry. Volume 21. Academic Press; Cambridge, MA, USA: 1966. pp. 113–286. PubMed

Augustin M.A., Chapman S.K., Davies D.M., Sykes A.G., Speck S.H., Margoliash E. Interaction of Cytochrome c with the Blue Copper Proteins, Plastocyanin and Azurin. J. Biol. Chem. 1983;258:6405–6409. doi: 10.1016/S0021-9258(18)32424-4. PubMed DOI

Bunkute E., Cummins C., Crofts F.J., Bunce G., Nabney I.T., Flower D.R. PIP-DB: The Protein Isoelectric Point Database. Bioinformatics. 2015;31:295–296. doi: 10.1093/bioinformatics/btu637. PubMed DOI

Slater J.C. Atomic Radii in Crystals. J. Chem. Phys. 1964;41:3199–3204. doi: 10.1063/1.1725697. DOI

Bonander N., Vänngård T., Tsai L.-C., Langer V., Nar H., Sjölin L. The Metal Site of Pseudomonas Aeruginosa Azurin, Revealed by a Crystal Structure Determination of the Co(II) Derivative and Co-EPR Spectroscopy. Proteins Struct. Funct. Bioinform. 1997;27:385–394. doi: 10.1002/(SICI)1097-0134(199703)27:3<385::AID-PROT6>3.0.CO;2-C. PubMed DOI

McLaughlin M.P., Retegan M., Bill E., Payne T.M., Shafaat H.S., Peña S., Sudhamsu J., Ensign A.A., Crane B.R., Neese F., et al. Azurin as a Protein Scaffold for a Low-Coordinate Nonheme Iron Site with a Small-Molecule Binding Pocket. J. Am. Chem. Soc. 2012;134:19746–19757. doi: 10.1021/ja308346b. PubMed DOI PMC

Zampino A.P., Masters F.M., Bladholm E.L., Panzner M.J., Berry S.M., Leeper T.C., Ziegler C.J. Mercury Metallation of the Copper Protein Azurin and Structural Insight into Possible Heavy Metal Reactivity. J. Inorg. Biochem. 2014;141:152–160. doi: 10.1016/j.jinorgbio.2014.09.003. PubMed DOI

Nar H., Huber R., Messerschmidt A., Filippou A.C., Barth M., Jaquinod M., van de Kamp M., Canters G.W. Characterization and Crystal Structure of Zinc Azurin, a by-Product of Heterologous Expression in Escherichia Coli of Pseudomonas Aeruginosa Copper Azurin. Eur. J. Biochem. 1992;205:1123–1129. doi: 10.1111/j.1432-1033.1992.tb16881.x. PubMed DOI

Bhagi-Damodaran A., Lu Y. The Periodic Table’s Impact on Bioinorganic Chemistry and Biology’s Selective Use of Metal Ions. In: Mingos D.M.P., editor. The Periodic Table II: Catalytic, Materials, Biological and Medical Applications. Springer International Publishing; Cham, Switzerland: 2019. pp. 153–173. (Structure and Bonding). PubMed PMC

Amdursky N., Sepunaru L., Raichlin S., Pecht I., Sheves M., Cahen D. Electron Transfer Proteins as Electronic Conductors: Significance of the Metal and Its Binding Site in the Blue Cu Protein, Azurin. Adv. Sci. 2015;2:1400026. doi: 10.1002/advs.201400026. PubMed DOI PMC

Lawrance G.A. Introduction to Coordination Chemistry. John Wiley & Sons; Hoboken, NJ, USA: 2013.

Maret W. Metalloproteomics, Metalloproteomes, and the Annotation of Metalloproteins. Met. Integr. Biometal Sci. 2010;2:117–125. doi: 10.1039/B915804A. PubMed DOI

Clementi E., Raimondi D.L., Reinhardt W.P. Atomic Screening Constants from SCF Functions. II. Atoms with 37 to 86 Electrons. J. Chem. Phys. 1967;47:1300–1307. doi: 10.1063/1.1712084. DOI

Pascher T., Karlsson B.G., Nordling M., Malmström B.G., Vänngård T. Reduction Potentials and Their pH Dependence in Site-Directed-Mutant Forms of Azurin from Pseudomonas Aeruginosa. Eur. J. Biochem. 1993;212:289–296. doi: 10.1111/j.1432-1033.1993.tb17661.x. PubMed DOI

Di Bilio A.J., Chang T.K., Malmström B.G., Gray H.B., Göran Karlsson B., Nordling M., Pascher T., Lundberg L.G. Electronic Absorption Spectra of M(II)(Met121X) Azurins (MCo, Ni, Cu; XLeu, Gly, Asp, Glu): Charge-Transfer Energies and Reduction Potentials. Inorganica Chim. Acta. 1992;198–200:145–148. doi: 10.1016/S0020-1693(00)92355-7. DOI

Garner D.K., Vaughan M.D., Hwang H.J., Savelieff M.G., Berry S.M., Honek J.F., Lu Y. Reduction Potential Tuning of the Blue Copper Center in Pseudomonas Aeruginosa Azurin by the Axial Methionine as Probed by Unnatural Amino Acids. J. Am. Chem. Soc. 2006;128:15608–15617. doi: 10.1021/ja062732i. PubMed DOI

Lowery M.D., Solomon E.I. Axial Ligand Bonding in Blue Copper Proteins. Inorganica Chim. Acta. 1992;198–200:233–243. doi: 10.1016/S0020-1693(00)92365-X. DOI

Marshall N.M., Garner D.K., Wilson T.D., Gao Y.-G., Robinson H., Nilges M.J., Lu Y. Rationally Tuning the Reduction Potential of a Single Cupredoxin beyond the Natural Range. Nature. 2009;462:113–116. doi: 10.1038/nature08551. PubMed DOI PMC

Winkler J.R., Gray H.B. Electron Flow through Metalloproteins. Chem. Rev. 2014;114:3369–3380. doi: 10.1021/cr4004715. PubMed DOI PMC

Voityuk A.A. Long-Range Electron Transfer in Biomolecules. Tunneling or Hopping? J. Phys. Chem. B. 2011;115:12202–12207. doi: 10.1021/jp2054876. PubMed DOI

Gray H.B., Winkler J.R. Long-Range Electron Transfer. Proc. Natl. Acad. Sci. USA. 2005;102:3534–3539. doi: 10.1073/pnas.0408029102. PubMed DOI PMC

Prytkova T.R., Kurnikov I.V., Beratan D.N. Coupling Coherence Distinguishes Structure Sensitivity in Protein Electron Transfer. Science. 2007;315:622–625. doi: 10.1126/science.1134862. PubMed DOI PMC

Takematsu K., Williamson H.R., Nikolovski P., Kaiser J.T., Sheng Y., Pospíšil P., Towrie M., Heyda J., Hollas D., Záliš S., et al. Two Tryptophans Are Better Than One in Accelerating Electron Flow through a Protein. ACS Cent. Sci. 2019;5:192–200. doi: 10.1021/acscentsci.8b00882. PubMed DOI PMC

Walden S.E., Wheeler R.A. Distinguishing Features of Indolyl Radical and Radical Cation: Implications for Tryptophan Radical Studies. J. Phys.Chem. 1996;100:1530–1535. doi: 10.1021/jp951838p. DOI