Crystal structure analysis of oxidized Pseudomonas aeruginosa azurin at pH 5·5 and pH 9·0

Routines for crystal orientation and the prediction of expected reflections which are part of the data collection software package MADNES for area-detector diffractometer systems in macromolecular crystallography are described. This package is designed to be area-detector-system independent. In addition to refining crystal cell lengths and angles, crystal orientation, crystal-to-detector distance, position of the primary beam on the detector, and rotation of the detector around the primary beam, the orientation routine also refines the effective mosaic spread of the crystal, the beam inclination angle μ and the detector tilt angle τ. A prealignment procedure is described for rapid rough orientation of the crystal. The routines are written in Fortran 77 in a modular way, so that they may be used independently of MADNES and each other.

The electron-transfer reactions of site-specific mutants of the blue copper protein azurin from Pseudomonas aeruginosa with its presumed physiological redox partners cytochrome c551 and nitrite reductase were investigated by temperature-jump and stopped-flow experiments. In the hydrophobic patch of azurin Met44 was replaced by Lys, and in the His35 patch His35 was replaced by Phe, Leu and Gln. Both patches were previously thought to be involved in electron transfer. 1H-NMR spectroscopy revealed only minor changes in the three-dimensional structure of the mutants compared to wild-type azurin. Observed changes in midpoint potentials could be attributed to electrostatic effects. The slow relaxation phase observed in temperature-jump experiments carried out on equilibrium mixtures of wild-type azurin and cytochrome c551 was definitively shown to be due to a conformational relaxation involving His35. Analysis of the kinetic data demonstrated the involvement of the hydrophobic but not the His35 patch of azurin in the electron transfer reactions with both cytochrome c551 and nitrite reductase.