Evidence for two distinct azurins in Alcaligenes xylosoxidans (NCIMB 11015): potential electron donors to nitrite reductase.
We have isolated two type 1 copper-containing proteins (M(r) approximately 13K) from Alcaligenes xylosoxidans (NCIMB 11015) grown under denitrifying conditions. Amino acid sequence analysis of these two proteins shows 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 and propose to call azurin II. Thus, NCIMB 11015 becomes the second system where two distinct azurins are found, the other being Methylomonas J (Ambler & Tobari, 1989). On isoelectric focusing, azurin I 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 than the azurin I sequence but is only 11% different than the azurin from Alcaligenes denitrificans NCTC 8582. Optical spectra for the two proteins 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 approximately 620 nm. The 291 nm shoulder normally ascribed to the hydrophobic nature of tryptophan 48 is clearly observed in azurin I but is missing in azurin 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 electron donors to the purified dissimilatory nitrite reductase of Alc. xylosoxidans in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)
Dodd
F E
FE
Molecular Biophysics Group, Daresbury Laboratory, Warrington, U.K.
Hasnain
S S
SS
Hunter
W N
WN
Abraham
Z H
ZH
Debenham
M
M
Kanzler
H
H
Eldridge
M
M
Eady
R R
RR
Ambler
R P
RP
Smith
B E
BE
D000421:Alcaligenes / Q000737:chemistry* / Q000201:enzymology; D000595:Amino Acid Sequence; D001400:Azurin / Q000737:chemistry* / Q000302:isolation & purification; D018360:Crystallography, X-Ray; D004583:Electrons; D008969:Molecular Sequence Data; D009572:Nitrite Reductases / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid
D001400:Azurin; D009572:Nitrite Reductases