Hydrogen exchange in Pseudomonas cytochrome c-551

Abstract

Hydrogen exchange rates were measured or estimated for 75 amide protons in in ferrocytochrome c-551 from Pseudomonas aeruginosa (82 residues total) at neutral pH and 300 K. Rate constants span at least eight orders of magnitude. Rate constants or limiting estimates were determined by a combination of methods relying upon 1H-NMR spectroscopy, including the direct observation in one- or two-dimensional spectra of the decrease in proton intensity for samples dissolved in deuterium oxide, or, in a few favorable cases, saturation transfer from the solvent protic water. The heme ligand residues and the thioether bridge residues were slowly exchanging backbone amides, but the slowest exchanging backbone amides were found in two clusters. One was composed of Ile-48 and Lys-49 in the last turn of what is termed the 40's helix in the protein. The second was composed of Leu-74, Ala-75, Lys-76 and Val-78 in the C-terminal alpha helix.