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 N₂O 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 are significantly less intense in preparations of 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 are 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 of the Cu_A-type sites.