Reversible acidic-alkaline transition of the carbon monoxide complex of cytochrome c peroxidase.
The Soret absorption band of the ferrous carbon monoxide (CO) complex of cytochrome c peroxidase exhibited a blue shift from 423.7 to 420 nm upon an increase in pH from 6.5 to 8.5. The spectral change was reversible with an isosbestic point at 422 nm. The pH dependence of this spectral change gave a sigmoidal curve fitted well to a theoretical curve of a cooperative release of two protons with a pK value of 7.5, indicating the existence of the acidic and alkaline forms of the ferrous CO enzyme. Upon irradiation of light flash (100 J of power and 30-microseconds), the heme-bound CO was readily dissociated in both acidic and alkaline forms with a quantum yield of approximately unity. On the other hand, the rate of recombination of the dissociated CO with the heme iron was significantly different between these two forms; the recombination rate constants were 1.1 X 10(3) and 3.0 X 10(4) M-1 S-1 at 25 degrees C for the acidic and alkaline forms, respectively. At intermediate pH values, kinetics of recombination were biphasic, consisting of the slow and fast processes with the appropriate rate constants mentioned above. When the fraction of the fast process was plotted against pH, the pH profile coincided with the spectrophotometric pH titration curve described above. Thus, it was concluded that the acidic and alkaline forms of the enzyme were responsible for the slow and fast processes, respectively. In infrared spectroscopy, the acidic form showed a narrow CO stretching band at 1922 cm-1 with a half-band width of 12.5 cm-1, while the alkaline form exhibited a broad CO-stretching band at 1948 cm-1 with a half-band width of 33 cm-1. Significance of these results are discussed in relation to the structure of the heme vicinity on the CO complex of cytochrome c peroxidase.
Iizuka
T
T
Makino
R
R
Ishimura
Y
Y
Yonetani
T
T
D002248:Carbon Monoxide / Q000378:metabolism*; D003578:Cytochrome-c Peroxidase / Q000378:metabolism* / Q000528:radiation effects; D005296:Ferrous Compounds / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D010544:Peroxidases / Q000378:metabolism*; D010782:Photolysis; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013053:Spectrophotometry; D013055:Spectrophotometry, Infrared
D005296:Ferrous Compounds; D002248:Carbon Monoxide; D010544:Peroxidases; D003578:Cytochrome-c Peroxidase