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 A 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 (pKa(red) = 7.3, pKa(ox) = 6.2), which lies in a crevice of the protein close to the copper binding site, involves a concomitant Pro36-Gly37 main-chain peptide bond flip. At the lower pH, the protonated imidazole N delta 1 of His35 forms a strong hydrogen bond with the carbonyl oxygen from Pro36, while at alkaline pH the deprotonated N delta 1 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.
Max Planck Institut für Biochemie, Martinsried bei München, Germany.
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D001312:Aurintricarboxylic Acid / Q000031:analogs & derivatives; D001559:Benzhydryl Compounds / Q000737:chemistry*; D003300:Copper / Q000737:chemistry; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa; D011994:Recombinant Proteins / Q000737:chemistry; D014961:X-Ray Diffraction
D001559:Benzhydryl Compounds; D011994:Recombinant Proteins; D001312:Aurintricarboxylic Acid; D003300:Copper; C017037:aurin