Electron Paramagnetic Resonance Study of the Interaction of Some Anionic Ligands with Oxidized Pseudomonas Cytochrome Oxidase*

Sodium fluoride induces changes in the heme dl components of both the frozen solution EPR and room temperature optical absorption spectra of oxidized Pseudomolurs cytochrome oxidase. The EPR resonances of the heme dl (iron-chlorin) moiety(ies) appear to undergo a rhombic to axial transition ( g = (2.52, 2.42, 1.73) 4 (2.54,2.54, 1.61)) with increasing concentrations of fluoride (0 3 0.1 M, pH 7.0) while the g values remain within the range usually attributed to low spin iron-porphyrin complexes (3.75 > g > 0.5). The axial spectrum which results is apparently low spin and suggests a degeneracy of two of the three iron d orbitals (d%, dyz, dw). The optical difference spectrum induced by sodium fluoride exhibits pronounced features at 475 and 646 nm which correspond to positions of heme dl optical absorption bands. The spectral alterations induced by fluoride are not due to a dimer 4 monomer transformation since gel chromatography of the oxidase in the absence and presence of sodium fluoride (0.1 M, pH 7.0) gives the same molecular weight. The heme c resonances of the oxidase are not altered substantially by the addition of sodium fluoride, and the axial ligation of the heme c moiety(ies) of the unperturbed oxidase is investigated using the observed g values. The interaction of oxidized Pseudomonas cytochrome oxidase with potassium cyanide causes distinct changes in both the heme dl and heme c components of the frozen solution EPR spectrum in which the former appears to retain its rhombic shape. Plausible modes of interaction of the enzyme with both fluoride and cyanide are discussed and, through the use of d orbital energy level diagrams, compared with energy levels calculated from published g values of iron-porphyrin and iron-chlorin moieties complexed with known axial ligands. Consistent differences between d orbital energy levels of iron-porphyrin and -chlorin (or chlorinlike) complexes are outlined.