SPECTROSCOPIC STUDIES OF THE COPPER SITES IN WILD-TYPE PSEUDOMONAS-STUTZERI N2O REDUCTASE AND IN AN INACTIVE PROTEIN ISOLATED FROM A MUTANT DEFICIENT IN COPPER-SITE BIOSYNTHESIS

Pseudomonas stutzeri N20 reductase is a complex multicopper enzyme (approximately 8 Cu ions/protein molecule). Two copper sites appear to be closely similar to the Cu(A)-type site in cytochrome c oxidase. but relatively little is known about the other copper sites in N2O reductase. In this paper circular dichroism, magnetic circular dichroism, and X-ray absorption and fluorescence spectroscopy have been used to further characterize the copper sites in native N2O reductase and in a "mutant" protein isolated from a strain deficient in the biosynthesis of the N2O reductase copper sites that contains only 2 copper ions/protein molecule. Both magnetic circular dichroism and X-ray absorption (Cu K-edge and EXAFS) data are consistent with the presence of (on average) one Cu(A)-type site per protein in the mutant N2O reductase. Comparisons of the near-infrared circular dichroism spectra of the oxidized native and "mutant" N2O reductases suggest that transitions at 7200 and 9500 cm-1 in the native enzyme are associated with copper sites other than the Cu(A)-type sites. To the best of our knowledge, these are the first electronic spectral features that can be attributed to non-Cu(A)-type sites in N2O reductase. The near-infrared bands arc significantly less intense in preparations or N2O reductase that display lower specific activities. Several electronic transitions are resolved in the circular and magnetic circular dichroism spectra of dithionite-reduced N2O reductase. Notably a near-infrared band is observed in the circular dichroism spectrum at 8200 cm-1. The data arc plausibly attributed to a highly covalent [Cu(II)-S-(cys) <--> Cu(I)-S.(cys)] site in the reduced enzyme. Copper removal from N2O reductase enhances the tryptophan fluorescence intensity about 3-fold; most of the quenching appears to be associated with occupation or the Cu(A)-type sites.