Catalytic and spectroscopic analysis of blue copper-containing nitrite reductase mutants altered in the environment of the type 2 copper centre: implications for substrate interaction.
The blue dissimilatory nitrite reductase (NiR) from Alcaligenes xylosoxidans is a trimer containing two types of Cu centre, three type 1 electron transfer centres and three type 2 centres. The latter have been implicated in the binding and reduction of nitrite. The Cu ion of the type 2 centre of the oxidized enzyme is ligated by three His residues, and additionally has a co-ordinated water molecule that is also hydrogen-bonded to the carboxyl of Asp(92) [Dodd, Van Beeumen, Eady and Hasnain (1998), J. Mol. Biol. 282, 369-382]. Two mutations of this residue have been made, one to a glutamic acid residue and a second to an asparagine residue; the effects of both mutations on the spectroscopic and catalytic properties of the enzyme have been analysed. EPR spectroscopy revealed that both mutants retained intact type 1 Cu centres with g( parallel)=2.12 (A( parallel)=0 mT) and g( perpendicular)=2.30 (A( perpendicular)=6.4 mT), which was consistent with their blue colour, but differed in their activities and in the spectroscopic properties of the type 2 centres. The D92E mutant had an altered geometry of its type 2 centre such that nitrite was no longer capable of binding to elicit changes in the EPR parameters of this centre. Accordingly, this mutation resulted in a form of NiR that had very low enzyme activity with the artificial electron donors reduced Methyl Viologen and sodium dithionite. As isolated, the EPR spectrum of the Asp(92)-->Asn (D92N) mutant showed no characteristic type 2 hyperfine lines. However, oxidation with iridium hexachloride partly restored a type 2 EPR signal, suggesting that type 2 copper is present in the enzyme but in a reduced, EPR-silent form. Like the Asp(92)-->Glu mutant, D92N had very low enzyme activities with either Methyl Viologen or dithionite. Remarkably, when the physiological electron donor reduced azurin I was used, both mutant proteins exhibited restoration of enzyme activity. The degree of restoration differed for the two mutants, with the D92N derivative exhibiting approx. 60% of the activity seen for the wild-type NiR. These findings suggest that on formation of an electron transfer complex with azurin, a conformational change in NiR occurs that returns the catalytic Cu centre to a functionally active state capable of binding and reducing nitrite.
Prudêncio
M
M
Department of Biological Chemistry, John Innes Centre, Colney Lane, Norwich NR4 7UH, U.K.
Eady
R R
RR
Sawers
G
G
D000421:Alcaligenes / Q000201:enzymology / Q000235:genetics*; D001400:Azurin / Q000737:chemistry; D002384:Catalysis; D003300:Copper / Q000032:analysis / Q000737:chemistry*; D003461:Crystallography; D004578:Electron Spin Resonance Spectroscopy; D006860:Hydrogen Bonding; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009572:Nitrite Reductases / Q000737:chemistry / Q000235:genetics*; D009573:Nitrites / Q000737:chemistry; D010084:Oxidation-Reduction; D013379:Substrate Specificity
D009573:Nitrites; D001400:Azurin; D003300:Copper; D009572:Nitrite Reductases