Accessibility of oxygen with respect to the heme pocket in horseradish peroxidase.
Oxygen and other molecules of similar size take part in a variety of protein reactions. Therefore, it is critical to understand how these small molecules penetrate the protein matrix. The protein system studied in this case is horseradish peroxidase (HRP). We have converted the native HRP into a phosphorescent analog by replacing the heme prosthetic group by Pd-mesoporphyrin. Oxygen readily quenches the phosphorescence of Pd porphyrins, and this can be used to characterize oxygen diffusion through the protein matrix. Our measurements indicate that solvent viscosity and pH modulate the accessibility of the heme pocket relative to small molecules. The binding of the substrate benzohydroxamic acid (BHA) to the protein drastically impedes oxygen access to the heme pocket. These results indicate that, first, the penetration of small molecules through the protein matrix is a function of protein dynamics, and second, there are specific pathways for the diffusion of these molecules. The effect of substrate and pH on protein dynamics has been investigated with the use of molecular dynamics calculations. We demonstrate that the model of a "fluctuating entry point," as suggested by Zwanzig (J Chem Phys 1992;97:3587-3589), properly describes the diffusion of oxygen through the protein matrix.
Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. mazdak@mail.med.upenn.edu
Mazdak
M
Khajehpour
Ivo
I
Rietveld
Sergei
S
Vinogradov
Ninad V
NV
Prabhu
Kim A
KA
Sharp
Jane M
JM
Vanderkooi
D001665:Binding Sites; D004058:Diffusion; D006418:Heme / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000378:metabolism; D006877:Hydroxamic Acids / Q000378:metabolism; D008163:Luminescent Measurements; D008652:Mesoporphyrins / Q000737:chemistry; D008958:Models, Molecular; D009281:Naphthalenes / Q000737:chemistry; D010100:Oxygen / Q000737:chemistry* / Q000378:metabolism; D010165:Palladium / Q000737:chemistry; D011485:Protein Binding; D012997:Solvents / Q000737:chemistry; D013057:Spectrum Analysis; D013464:Sulfuric Acids / Q000737:chemistry; D014783:Viscosity
D006877:Hydroxamic Acids; D008652:Mesoporphyrins; D009281:Naphthalenes; D012997:Solvents; D013464:Sulfuric Acids; D006418:Heme; C031468:mesoporphyrin IX; D010165:Palladium; C072598:6-bromo-2-naphthyl sulfate; D006735:Horseradish Peroxidase; C006696:benzohydroxamic acid; D010100:Oxygen