| Abstract: | The 1H NMR spectra of ferri- and ferro-cytochrome c-550 from Paracoccus denitrificans (ATCC 13543) have been investigated at 300 MHz. The ferri-cytochrome c-550 shows hyperfine-shifted heme methyl resonances at 29.90, 29.10, 16.70, and 12.95 ppm and a ligand methionyl methyl resonance at -15.80 ppm (pH 8 and 23 degrees C). Four pH-linked structural transitions were detected in spectra taken as a function of pH. The transitions have been interpreted as loss of the histidine heme ligand (pK less than or equal to 3), ionization of a buried heme propionate (pK = 6.3 +/- 0.2), displacement of the methionine heme ligand by a lysyl amino group (pK congruent to 10.5), and loss of the lysyl ligand (pK greater than or equal to 11.3). The temperature behavior of hyperfine-shifted resonances was determined. Two heme methyl resonances (at 16.70 and 12.95 ppm) showed downfield hyperfine shifts with increasing temperature. The cyanoferricytochrome had methyl resonances at 23.3, 20.1, and 19.4 ppm. NMR spectroscopy did not detect the formation of a complex with azide. The second-order rate constant for electron transfer between ferric and ferrous forms was determined to be 1.6 X 10(4) M-1 s-1. Heme proton resonances were assigned in both oxidation states by cross-saturation and nuclear Overhauser enhancement experiments. Spin-coupling patterns in the aromatic region of the ferro-cytochrome spectrum were investigated. |
