CRYSTAL-STRUCTURE ANALYSIS OF OXIDIZED PSEUDOMONAS-AERUGINOSA AZURIN AT PH 5.5 AND PH 9.0 - A PH-INDUCED CONFORMATIONAL TRANSITION INVOLVES A PEPTIDE-BOND FLIP

The X-ray crystal structure of recombinant wild-type azurin from Pseudomonas aeruginosa was determined by difference Fourier techniques using phases derived from the structure of the mutant His35Leu. Two data sets were collected from a single crystal of oxidized azurin soaked in mother liquor buffered at pH 5·5 and pH 9·0, respectively. Both data sets extend to 1·93 Å resolution. The two pH forms were refined independently to crystallographic R-factors of 17·6% (pH 5·5) and 17·5% (pH 9·0). The conformational transition previously attributed to the protonation/deprotonation of residue His35 (pKared = 7·3, pKaox = 6·2), which lies in a crevice of the protein close to the copper binding site, involves a concomitant Pro36Gly37 main-chain peptide bond flip. At the lower pH, the protonated imidazole Nσ1 of His35 forms a strong hydrogen bond with the carbonyl oxygen from Pro36, while at alkaline pH the deprotonated Nσ1 acts as an acceptor of a weak hydrogen bond from HN Gly37. The structure of the remainder of the azurin molecule, including the copper binding site, is not significantly affected by this transition. © 1991.