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 syst em for the copper-containing nitrite reductase from a denitrifying var iant of Rhodobacter sphaeroides. Typical yields of wild-type protein a re 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/elec tron 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 perturbatio n 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 meas ured 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 cent er in the absence of nitrite is not energetically favored. The mutatio n 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 reminisce nt 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 unchan ged from these of the blue-green native Type 1 center. His(287) is a r esidue in the Type 2 region whose imidazole ring was thought to hydrog en 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.