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.
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Max Planck Institut für Biochemie, Martinsried bei München, Germany.
Messerschmidt
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A
Huber
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van de Kamp
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M
Canters
G W
GW
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