Azurin
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| Azurin | |||||||
|---|---|---|---|---|---|---|---|
 Azurin tetramer, Pseudomonas aeruginosa | |||||||
| Identifiers | |||||||
| Symbol | Copper-bind | ||||||
| Pfam | PF00127 | ||||||
| SCOP2 | 5azu / SCOPe / SUPFAM | ||||||
| CDD | cd13922 | ||||||
| |||||||
Azurin is a small, periplasmic, bacterial blue copper protein found in Pseudomonas, Bordetella, or Alcaligenes bacteria. Azurin moderates single-electron transfer between enzymes associated with the cytochrome chain by undergoing oxidation-reduction between Cu(I) and Cu(II). Each monomer of an azurin tetramer has a molecular weight of approximately 14kDa, contains a single copper atom, is intensively blue, and has a fluorescence emission band centered at 308 nm.
Azurins and pseudoazurins participate in the denitrification processes in bacteria.,[1] including the gram-negative bacteria Pseudomonas aeruginosa, by interacting with cytochrome c551. Azurin from P aeruginosa is a type I blue copper protein (cupredoxin), while cytochrome c551 (9 kDa) is a haem-containing cytochrome. Azurin possesses a relatively large hydrophobic patch close to the active site, and two residues in this hydrophobic patch, Met-44 and Met-64, are believed to be involved in its interaction with the redox partners cytochrome c551 and nitrite reductase.[2]
Although unrelated to its electron-transfer property, azurin has been found to have anticancer properties through its interaction with tumor-suppressor protein p53.
In its oxidized form, azurin (Cu2+Az) receives an electron from its redox partner and is reduced according to the following reaction:
- Cu2+Az + e− → Cu+Az
The redox potential is 310 mV.[3]
The highly interconnected beta-sheet structure of azurin is strongly coupled with its electron-transfer center (the copper-binding side).[4] Considerable experimental evidence exists to suggest that hydrogen bonds play a role in the long-distance electron transfer mechanism of azurin. Taken together, these observations suggest that electrons tunnel through the protein along its polypeptide and hydrogen bonds, making azurin a useful model system for studying long-range, intraprotein electron transfer (LRET).[4]
Protein structure
Azurin is a monomeric protein that weighs approximately 14 kDa and is composed of 128 amino acids forming eight beta-strands arranged in a beta-barrel formation.[5] The strands are connected by turns and a single alpha-helical insertion.[5] A single-atom copper binding site is located about 7 Å below each monomer's surface towards its northern end; the copper atom that inhabits it is coordinated by five ligands surrounded by an extensive hydrophobic patch.[6]
The three equatorial copper ligands are composed of a thiolate (Cys112) and two imidazoles (His46, His117), and the carbonyl oxygen atoms of Gly45 and Met121 serve as the two weak axial ligands.[5] With the exception of Gly45, the copper-binding configuration above is common to the structures of all blue type 1 copper-binding proteins determined thus far.[7] Once coordinated, the ligand-metal complex assumes a distorted, trigonal bi-pyramidal geometry that stabilizes the cuprous (Cu(I)) reduced state of the protein relative to the cupric (Cu(II)) oxidized state.[5] Structurally imposed backbonding between the copper d orbitals and its ligand p orbitals may further stabilize the cuprous state.[8]
Existing structural information about azurin has largely been derived from X-ray crystallography studies of single-site mutated forms of the protein. Notable structural features elucidated by crystallography include the beta-sandwich motif formed from eight interlocking beta strands,[5] as well as an alpha-helical segment outside the barrel linking beta-sheets 4 and 5.[5]
Although the Cu(I)/Cu(II) redox potential is typically higher for azurin than most other copper complexes, structural studies in which Met121 (one of azurin's equatorial copper-coordinating ligands) is replaced have demonstrated that the absence of a thiolate copper ligand does not preclude high reduction potentials, as large hydrophobic residues in position 121 also raise the redox potential of the copper atom.[8] Thus, the higher redox potentials have been attributed to the exclusion of water from the metal-binding site, a condition augmented by the presence of bulky hydrophobic residues.[8]
Conversely, negatively charged residues lower the redox potential, since they stabilize the more positively charged cupric form of the copper ion.[8]
Biological function
When expressed in nitrogen-fixing organisms, azurin serves as the electron donor to nitrite reductase, an enzyme in the denitrification pathway of the nitrogen cycle.[9]
Azurins support oxidative deamination of primary amines by passing electrons from aromatic amine dehydrogenase to cytochrome oxidase, as well as from some c-type cytochromes to nitrite reductases.[10]