Azurin

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 enzyme along its polypeptide and hydrogen bonds, making azurin is a useful model system for studying long-range, intraprotein electron transfer (LRET).[4]

The copper binding domain of azurin rendered in PyMol, with four of the five copper-binding ligands labeled. The distance (in angstroms) from the copper atom to each individual ligand has been recorded.

Azurin is a tetrameric protein.[5] Each monomer weighs approximately 14kDa 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 7A 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, His 117), 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]

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]

Azurin has garnered significant attention as a potential therapeutic for various diseases, including cancer.[11] In vivo, it has been demonstrated to induce regression of human melanoma and breast cancer tissue with minimal toxic effects to the organism.[11]

Azurin enters preferentially into cancer cells via the p28 domain of the enzyme, which roughly corresponds to the extended alpha-helical region of the enzyme.[11] In cancer cells, azurin complexes with p53, stabilizing it and preventing association with E3 uiquitin ligases, which would otherwise bind and mark the protein for destruction.[12] Four azurin molecules bind each p53 monomer with high affinity.[12] The p53/azurin complex travels to the nucleus, where p53 upregulates the transcription of proapoptotic genes Bax and Noxa.[12] P53 also activates the expression of cell-cycle inhibitors, preventing tumor cells from progressing beyond the G1 or S phase.[12] Although this pathway plays a significant role in azurin’s anticancer activity, the details of the interaction between azurin and p53 are not well understood.

A phase I clinical trial in the United States demonstrated both partial and complete tumor regression effects in fifteen stage IV cancer patients treated with the p28 amino-acid fragment of azurin.[13] Another phase I trial with the p28 fragment demonstrated azurin’s therapeutic effects against pediatric patients with brain tumors; subsequently, the USFDA approved the designation of p28 as an orphan drug for glioma.[14]

Azurin’s other domains may also exhibit strong anticancer activity by binding to cell surface receptor tyrosine kinases such as EphB2 receptors, which induce angiogenesis in cancer cells.[14] This is another mechanism by which azurin has been proposed to exhibit its therapeutic effects.

• Plastocyanin family of copper-binding proteins