Spectroscopic and electrochemical properties of two azurins (Az-iso1 and Az-iso2) from the obligate methylotroph Methylomonas sp. strain J and the structure of novel Az-iso2

Methylomonas

sp. strain J gives rise to two azurins (Az-iso1 and Az-iso2) with methylamine dehydrogenase (MADH-Mj). The intense blue bands characteristic of Az-iso1 and Az-iso2 are observed at 621 and 616 nm in the visible absorption spectra respectively, being revealed at 620−630 nm in those of usual azurins. The EPR signal of Az-iso1, similar to usual azurins, shows axial symmetry, while the axial EPR signal of Az-iso2 involves a slightly rhombic character. The half-wave potentials (E _1/2) of the two azurins and the intermolecular electron-transfer rate constants (k _ET) from MADH-Mj to each azurin were determined by cyclic voltammetry. The E _1/2 values of Az-iso1 and Az-iso2 are +321 and +278 mV vs NHE at pH 7.0, respectively. The k _ET value of Az-iso2 is larger than that of Az-iso1 by a factor of 5. However, the electron-transfer rate of Az-iso2 is interestingly slower than those of the azurins from a denitrifying bacterium, Alcaligenes xylosoxidans NCIB 11015, and the amicyanin from a different methylotroph, Methylobacterium extorquens AM1. The structure of Az-iso2 has been determined and refined against 1.6 Å X-ray diffraction data. The whole structure of Az-iso2 is quite similar to those of azurins reported already. The Cu(II) site of Az-iso2 is a distorted trigonal bipyramidal geometry like those of other azurins, but some of the Cu-ligand distances and ligand-Cu-ligand bond angle parameters are slightly different. These findings suggest that Az-iso2 is a novel azurin and perhaps functions as an electron acceptor for MADH.