EVIDENCE FOR 2 DISTINCT AZURINS IN ALCALIGENES-XYLOSOXIDANS (NCIMB-11015) - POTENTIAL ELECTRON-DONORS TO NITRITE REDUCTASE

We have isolated two type 1 copper-containing proteins (M(r) similar t o 13K) from Alcaligenes xylosoxidans (NCIMB 11015) grown under denitri fying conditions. Amino acid sequence analysis of these two proteins s hows one to be the previously identified azurin (Ambler, 1971), which we shall call azurin I, and the other to be a related, but previously undescribed, blue copper protein which we show to also be an azurin an d propose to call azurin II. Thus, NCIMB 11015 becomes the second syst em where two distinct azurins are found, the other being Methylomonas J (Ambler & Tobari, 1989). On isoelectric focusing, azurin 1 migrates very similarly to the previously identified azurin from this organism while azurin II migrates similarly to azurin purified from Alcaligenes denitrificans NCTC 8582. The sequence of azurin II is 33% different t han the azurin I sequence but is only 11% different than the azurin fr om Alcaligenes denitrificans NCTC 8582. Optical spectra for the two pr oteins are very similar with epsilon(mM) values of 6.27 and 5.73 mM(-1 ) cm(-1) for azurin I and II, respectively, at lambda(max) similar to 620 nm. The 291 nm shoulder normally ascribed to the hydrophobic natur e of tryptophan 48 is clearly observed in azurin I but is missing in a zurin II. Amino acid analysis confirms that this tryptophan is missing in azurin II. Azurin I and azurin II show essentially the same redox potential of 305 +/- 10 mV at pH 7.5 and are equally effective electro n donors to the purified dissimilatory nitrite reductase of Alc. xylos oxidans in vitro, The two azurins crystallized distinctly; azurin I cr ystallizes as thin needles while azurin II grows as blue elongate rect angular prisms with the tetragonal space group P4(1)22 and unit cell p arameters a = b = 52.65 Angstrom, c = 100.63 Angstrom. The structure o f azurin II has been solved and is currently being refined to 1.9 Angs trom resolution.