Perturbation of Pseudomonas cytochrome oxidase by guanidine hydrochloride to detect differential stabilization of the heme d1 and heme c moieties.
The optical properties of Pseudomonas cytochrome oxidase (ferrocytochrome-c:oxygen oxidoreductase, EC 1.9.3.2) were monitored as a function of guanidine hydrochloride (Gdn X HCl) concentration to probe for differential stabilization of its prosthetic groups, heme d1 and heme c. The protein fluorescence intensity increased with the Gdn X HCl concentration, revealing two transitions, a sharp one between 1.3 and 1.5 M Gdn X HCl, and a second less well defined extending from 2.5 to 4.5 M. Only the transition at the lower Gdn X HCl concentrations was present in titrations followed using the emission maxima. The spectral maximum for native Pseudomonas cytochrome oxidase was at approx. 335 nm and shifted to approx. 350 nm above 2 M Gdn X HCl. The heme d1 absorbance at 638 nm decreased with increasing [Gdn X HCl], giving a transition at 1.3-1.5 M, and no transition up to 4 M Gdn X HCl when the heme c was monitored at 525 nm. Along with the decrease at 638 nm, an absorption band appeared at 681 nm, suggesting heme d1 release into solution. Fluorescence titration of heme d1-depleted enzyme, prepared by gel filtration, showed a single transition similar to the transition occurring in the intact enzyme at high Gdn X HCl concentrations. Circular dichroism spectra revealed clearly distinguishable transitions for the heme d1 and heme c near 1.5 and 3.0 M Gdn X HCl, respectively. These results suggest that the two hemes are in regions of the protein with different stabilities which may represent distinct structural domains.
D002850:Chromatography, Gel; D002942:Circular Dichroism; D003576:Electron Transport Complex IV / Q000378:metabolism*; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology*; D006418:Heme / Q000031:analogs & derivatives* / Q000378:metabolism; D046911:Macromolecular Substances; D011549:Pseudomonas / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
D006146:Guanidines; D046911:Macromolecular Substances; C031690:heme C; D006418:Heme; C029400:heme d1; D003576:Electron Transport Complex IV; D019791:Guanidine