Alcaligenes xylosoxidans dissimilatory nitrite reductase: alanine substitution of the surface-exposed histidine 139l ligand of the type 1 copper center prevents electron transfer to the catalytic center.
Nitrite reductase of Alcaligenes xylosoxidans contains three blue type 1 copper centers with a function in electron transfer and three catalytic type 2 copper centers. The mutation H139A, in which the solvent-exposed histidine ligand of the type 1 copper ion was changed to alanine, resulted in the formation of a colorless protein containing 4.4 Cu atoms per trimer. The enzyme was inactive with reduced azurin as the electron donor, and in contrast to the wild-type enzyme, no EPR features assignable to type 1 copper centers were observed. Instead, the EPR spectrum of the H139A enzyme, with parameters of g(1) = 2.347 and A(1) = 10 mT, was typical of type 2 copper centers. On the addition of nitrite, the EPR features developed spectral features with increased rhombicity, with g(1) = 2.29 and A(1) = 11 mT, arising from the type 2 catalytic site. As assessed by visible spectroscopy, ferricyanide (E degree = +430 mV) was unable to oxidize the H139A enzyme, and this required a 30-fold excess of K(2)IrCl(6) (E degree = +867 mV). Oxidation resulted in the EPR spectrum developing additional axial features with g(1) = 2.20 and A(1) = 9.5 mT, typical of type 1 copper centers. The oxidized enzyme after separation from the excess of K(2)IrCl(6) by gel filtration was a blue-green color with absorbance maxima at 618 and 420 nm. The instability of the protein prevented the precise determination of the midpoint potential, but these properties indicate that it is in the range 700-800 mV, an increase of at least approximately 470 mV compared with the native enzyme. This high potential, which is consistent with a trigonal planar geometry of the Cu ion, effectively prevents azurin-mediated electron transfer from the type 1 center to the catalytic type 2 Cu site. However, with dithionite as reductant, 20% of the activity of the wild-type enzyme was observed, indicating that the direct reduction of the catalytic site by dithionite can occur. When CuSO(4) was added to the crude extract before isolation of the enzyme, the Cu content of the purified H139A enzyme increased to 5.7 Cu atoms per trimer. The enzyme remained colorless, and the activity with dithionite as a donor was not significantly increased. The additional copper in such preparations was associated with an axial type 2 Cu EPR signal with g(1) = 2.226 and A(1) = 18 mT, and which were not changed by the addition of nitrite, consistent with the activity data.
Prudêncio
Miguel
M
Departments of Biological Chemistry and Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, U.K.
Sawers
Gary
G
Fairhurst
Shirley A
SA
Yousafzai
Faridoon K
FK
Eady
Robert R
RR
D000409:Alanine / Q000737:chemistry*; D000421:Alcaligenes / Q000201:enzymology*; D020134:Catalytic Domain; D003300:Copper / Q000378:metabolism*; D003580:Cytochromes; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry*; D008024:Ligands; D009572:Nitrite Reductases / Q000737:chemistry / Q000378:metabolism*
D003580:Cytochromes; D008024:Ligands; D006639:Histidine; D003300:Copper; D009572:Nitrite Reductases; C028123:nitrite reductase (NO-forming); D003576:Electron Transport Complex IV; D000409:Alanine