High-potential C112D/M121X (X = M, E, H, L) Pseudomonas aeruginosa azurins

Abstract

Site-directed mutagenesis of Pseudomonas aeruginosa azurin C112D at the M121 position has afforded a series of proteins with elevated Cu(II/I) reduction potentials relative to the Cu(II) aquo ion. The high potential and low axial hyperfine splitting (Cu(II) electron paramagnetic resonance A( parallel)) of the C112D/M121L protein are remarkably similar to features normally associated with type 1 copper centers.

Figure 1

PyMol rendering of the active site of C112D azurin (PDBID: 1AG0).

Figure 2

Electronic absorption spectra of Cu(II) (A) and Co(II) (B) C112DM121X azurins in 10 mM sodium phosphate pH 7.0. X = M (black), L (green), E (red), H (blue).

Figure 3

77 K X-band EPR spectra of Cu(II) C112DM121X azurins in 10 mM sodium phosphate pH 7.0. X = M (black), L (green), E (red), H (blue).

Figure 4

Electrochemistry of C112D/M121X azurins on SAM-modified Au electrodes. C112D (black) and C112D/M121L (green) potentials were measured by CV in 10 mM sodium phosphate pH 7.0 at a scan rate of 50 mV/s referenced to Ag/AgCl in saturated KCl (197 mV vs NHE). Azurin C112D/M121E (red) was measured by SWV in 10 mM NaP_i with a square wave frequency of 8 Hz.