Activation of the cytochrome c peroxidase of Pseudomonas aeruginosa. The role of a heme-linked protein loop: a mutagenesis study.
Mutagenesis studies have been used to investigate the role of a heme ligand containing protein loop (67-79) in the activation of di-heme peroxidases. Two mutant forms of the cytochrome c peroxidase of Pseudomonas aeruginosa have been produced. One mutant (loop mutant) is devoid of the protein loop and the other (H71G) contains a non-ligating Gly at the normal histidine ligand site. Spectroscopic data show that in both mutants the distal histidine ligand of the peroxidatic heme in the un-activated enzyme is lost or is exchangeable. The un-activated H71G and loop mutants show, respectively, 75% and 10% of turnover activity of the wild-type enzyme in the activated form, in the presence of hydrogen peroxide and the physiological electron donor cytochrome c(551). Both mutant proteins show the presence of constitutive reactivity with peroxide in the normally inactive, fully oxidised, form of the enzyme and produce a radical intermediate. The radical product of the constitutive peroxide reaction appears to be located at different sites in the two mutant proteins. These results show that the loss of the histidine ligand from the peroxidatic heme is, in itself, sufficient to produce peroxidatic activity by providing a peroxide binding site and that the formation of radical intermediates is very sensitive to changes in protein structure. Overall, these data are consistent with a major role for the protein loop 67-79 in the activation of di-heme peroxidases and suggest a "charge hopping" mechanism may be operative in the process of intra-molecular electron transfer.
Hsiao
Hsi-Chen
HC
Centre for Metalloprotein Spectroscopy and Biology, School of Biological Sciences, University of Auckland, Auckland, New Zealand.
Boycheva
Svetlana
S
Watmough
Nicholas J
NJ
Brittain
Thomas
T
D003578:Cytochrome-c Peroxidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation / Q000235:genetics; D005998:Glycine / Q000235:genetics; D006418:Heme / Q000378:metabolism* / Q000502:physiology; D006420:Hemeproteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006639:Histidine / Q000235:genetics; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics
D006420:Hemeproteins; D008024:Ligands; D011994:Recombinant Proteins; D006418:Heme; D006639:Histidine; D003578:Cytochrome-c Peroxidase; D005998:Glycine