Alcaligenes xylosoxidans Dissimilatory Nitrite Reductase: Alanine Substitution of the Surface-Exposed Histidine 139 Ligand of the Type 1 Copper Center Prevents Electron Transfer to the Catalytic Center † Miguel Prude ˆncio, ‡,§ Gary Sawers, ‡ Shirley A. Fairhurst, | Faridoon K. Yousafzai, | and Robert R. Eady* ,| Departments of Biological Chemistry and Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, U.K. ReceiVed October 22, 2001; ReVised Manuscript ReceiVed January 14, 2002 ABSTRACT: 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° )+430 mV) was unable to oxidize the H139A enzyme, and this required a 30-fold excess of K 2 IrCl 6 (E° )+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 ∼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. The process of denitrification, in which nitrate undergoes stepwise reduction to the gaseous products nitrous oxide and dinitrogen, forms one of the main branches of the global nitrogen cycle. In addition to its importance in microbial bioenergetics, further impetus for studying denitrification is provided by its environmental impact in generating N 2 O as a potent greenhouse gas and as a process which removes polluting nitrate from groundwater (1-3). The conversion of nitrite to nitric oxide, catalyzed by dissimilatory nitrite reductases, is a key step in denitrification since it is the point at which fixed nitrogen in the soil is converted to a gaseous product, which can result in significant losses of nitrogen to the atmosphere. Two classes of periplasmic nitrite reductases (NiR) 1 have been isolated from denitrifying microorganisms. One class of enzymes has cd 1 heme as the prosthetic group, while the second class includes enzymes that contain only copper. The copper-based NiRs are divided into two subclasses based on their color, being either blue or green (3). Crystallographic structures of two green NiRs, the enzymes from Achromo- bacter cycloclastes (AcNiR) (4-6) and Alcaligenes faecalis (AfNiR) (7), and the blue NiR from Alcaligenes xylosoxidans (AxNiR) (8-10) have been reported. These studies revealed that although these enzymes are trimers with very similar overall structures, they exhibit striking differences in surface † This work was supported by the Biotechnology and Biological Sciences Research Council as part of the competitive strategic grant to the John Innes Centre and by a PRAXIS XXl studentship (BD 5451/ 95) to M.P. * To whom correspondence should be addressed. Tel: +44 1603 450728. Fax: +44 1603 450018. E-mail: robert.eady@bbsrc.ac.uk. ‡ Department of Molecular Microbiology, John Innes Centre. § Present address: Leiden Institute of Chemistry, Gorlaeus Labora- tories, Leiden University, 2300 RA Leiden, The Netherlands. | Department of Biological Chemistry, John Innes Centre. 1 Abbreviations: NiR, nitrite reductase; AxNiR, nitrite reductase from Alcaligenes xylosoxidans; AcNiR, nitrite reductase from Achromobacter cycloclastes; AfNiR, nitrite reductase from Alcaligenes faecalis; RsNiR, nitrite reductase from Rhodopseudomonas sphaeroides; EPR, electron paramagnetic resonance; ENDOR, electron nuclear double resonance; EXAFS, extended X-ray absorption fine structure; MV, methyl violo- gen. 3430 Biochemistry 2002, 41, 3430-3438 10.1021/bi011955c CCC: $22.00 © 2002 American Chemical Society Published on Web 02/12/2002