Premium
Flexibility in Proteins: Tuning the Sensitivity to O 2 Diffusion by Varying the Lifetime of a Phosphorescent Sensor in Horseradish Peroxidase ¶
Author(s) -
Nibbs Janna,
Vinogradov Sergei A.,
Vanderkool Jane M.,
Zelent Bogumil
Publication year - 2004
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/j.1751-1097.2004.tb00046.x
Subject(s) - phosphorescence , chemistry , quenching (fluorescence) , horseradish peroxidase , quantum yield , photochemistry , peroxidase , fluorescence , oxygen , organic chemistry , enzyme , physics , quantum mechanics
The heme in horseradish peroxidase (HRP) was replaced by phosphorescent Pt‐mesoporphyrin IX (PtMP), which acted as a phosphorescent marker of oxygen quenching and allowed comparison with another probe, Pd‐mesoporphyrin IX (Khajehpour et al. (2003) Proteins 53, 656–666). Benzohydroxamic acid (BHA), a competitive inhibitor of the enzyme, was also used to monitor its effects on phosphorescence quenching. With the addition of BHA, in the presence of oxygen, the phosphorescence intensity of the protein increased. In contrast, the addition of BHA, in the absence of oxygen, reduced the phosphorescence intensity of the protein. K d = 18 μM when BHA binds to PtMP‐HRP. The effect of BHA can be explained by two factors: (1) BHA reduces the accessibility of O 2 to the protein interior and (2) BHA itself quenches the phosphorescence. Consistent with this, the oxygen quenching of the phosphorescence of PtMP‐HRP gave a quenching constant of k q = 234 mm Hg −1 s −1 in the absence of BHA and k q = 28.7 mm Hg −1 s −1 in the presence of BHA. The quenching rate of BHA is 4000 s −1 . The relative quantum yield of the phosphorescence of the Pt derivative is about six times that of the Pd derivative, whereas the phosphorescence lifetime is approximately eight times shorter. The high quantum yield and suitable lifetime make Pt‐porphyrins appropriate as sensors of O 2 diffusion and flexibility in heme proteins.