Novel Biological Copper Proteins through Artion Addition to the Mutant Metl21Gly of Pseudomonas aeruginosa Azurin

摘要

Blue cupper proteins arc widely distributed in nature, where they function as electron-transfer mediators in important biochemical processes such as photosynthesis and metabolism. The unique spectrochemical properties of these proteins have long attracted the attention of biomorganic chemists. For proteins in the oxidized state [copperun]. these characteristics include an intense absorption band centered around 600 nm (epsilon=2000 - 6000 M ~(-1)cm~(-1)), an EPR spectrum with an unusually small copper hyperfine splitting (I=32. A <= 70 x 10~(-4) cm~(-1)), and anomalously high redox potentials for the Cu~(II) Cu~I couple [180 - 680 mV versus the normal hydrogen electrode (NHE)j. Crystallographic analysis has revealed that the copper(n) ion in these proteins is coordinated to two histidine residues and one cysteine residue in an approximately trigonal planar geometry. Besides these three strong interactions, weaker axial interactions between the copper ion and other lig-ands are generally present. For example, in uzurin and plasto-eyanin, the thioether sulfur atom of a methionine residue interacts with the copper center, while in stellacyanm. a glutamine carboxamide oxygen is thought to coordinate to the metal. The nature of the axial interaction isbelieved to significantly influence the spectrochemical characteristics of metal centers of the blue copper proteins. To probe the effect of various axial ligands on the spectroscopic and electrochemical properties of these copper sites, we have prepared by site-directed mutagene-sis a variant of Pseudomonas aeruginosa azurin. Metl21Gly. in which the weakly coordinating (R_s_Cu = 3.1 A) thioether side chain of Metl21 is replaced by a hydrogen atom. This creates a cavity that can be occupied by exogenous ligands. In this communication we report the preparation and characterization of unique biological copper species, obtained through addition of the anions N_3~-, SCN~-, and CN~- to Metl21Gly azurin. Their spectroscopic properties arc distinct from those of any known naturally occurring copper proteins.