Spectroscopic, kinetic, and electrochemical characterization of heterologously expressed wild-type and mutant forms of copper-containing nitrite reductase from Rhodobacter sphaeroides 2.4.3.
We report the development of a high-yield heterologous expression system for the copper-containing nitrite reductase from a denitrifying variant of Rhodobacter sphaeroides. Typical yields of wild-type protein are 20 mg L-1, which can be fully loaded with copper. Nitrite reductase contains an unusual blue-green Type 1 copper center with a redox/electron transfer function and a nearby Type 2 center where nitrite binds and is reduced to nitric oxide. The wild-type enzyme was characterized by: (1) its blue-green Type 1 optical spectrum; (2) its EPR spectrum showing rhombic character to its Type 1 center and nitrite perturbation to its Type 2 center; (3) its 247-mV Type 1 midpoint potential which is low relative to other Type 1 centers; and (4) its kinetics as measured by both steady-state and stopped-flow methods. The Type 2 copper reduction potential as monitored by EPR in the absence of nitrite was below 200 mV so that reduction of the Type 2 center by the Type 1 center in the absence of nitrite is not energetically favored. The mutation M182T in which the methionine ligand of Type 1 copper was changed to a threonine resulted in a blue rather than blue-green Type 1 center, a midpoint potential that increased by more than 100 mV above that of the wild-type Type 1 center, and a somewhat reduced nitrite reductase activity. The blue color and midpoint potential of M182T are reminiscent of plastocyanin, but the Type 1 cupric HOMO ground-state electronic g value and copper hyperfine properties of M182T (as well as cysteine and histidine ENDOR hyperfine properties; see next paper) were unchanged from those of the blue-green native Type 1 center. His287 is a residue in the Type 2 region whose imidazole ring was thought to hydrogen bond to the Type 2 axial ligand but not directly to Type 2 copper. The mutation H287E resulted in a 100-fold loss of enzyme activity and a Type 2 EPR spectrum (as well as ENDOR spectra; see next paper) which were no longer sensitive to the presence of nitrite.
Olesen
K
K
Department of Chemistry, Center for Biophysics and Biochemistry, University at Albany, SUNY, New York 12222, USA.
Veselov
A
A
Zhao
Y
Y
Wang
Y
Y
Danner
B
B
Scholes
C P
CP
Shapleigh
J P
JP
D003300:Copper / Q000378:metabolism*; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy; D005822:Genetic Vectors / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D016254:Mutagenesis, Insertional; D009572:Nitrite Reductases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D010084:Oxidation-Reduction; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000235:genetics; D013053:Spectrophotometry
D003300:Copper; D009572:Nitrite Reductases; C052695:nitrite reductase, copper-containing