Use of bilirubin oxidase for probing chromophore topography in tetrapyrrole proteins.
Bilirubin oxidase has been used to probe the surface topography of phycocyanins (C-phycocyanin and phycocyanin-645), peridinin-chlorophyll a-protein and phytochrome. The enzyme catalyzes oxidation of the tetrapyrrolic chromophores in these proteins. Relative rates of oxidation were 78.0 X 10(-6) s-1 (monitored at 617 nm) and 58.0 X 10(-6) s-1 (592 nm) for C-phycocyanin, 43.0 X 10(-6) s-1 for phycocyanin-645, 0.3 X 10(-6) s-1 (at 671 nm) and 1.3 X 10(-6) s-1 (at 480 nm) for peridinin-chlorophyll a-protein. The relative rate of free chlorophyllin a was 2.8 X 10(4) s-1 whereas upon binding to human serum albumin its rate of oxidation was reduced to 3.3 X 10(-3) s-1. Relative rates for the oxidation of Pr and Pfr forms of phytochrome were 2.9 and 19.5 s-1, respectively, which are consistent with earlier finding [( 1984) Plant Physiol. 74, 755-758] that indicated a preferential exposure of tetrapyrrolic chromophore in the Pfr form. In general, kcat/Km values derived from the Lineweaver-Burk plots followed the same trend as the relative rates of oxidation. For example, the kcat/Km for the free chlorophyllin a was 2.8 X 10(6) M-1 s-1 but it was only 1.1 M-1s-1 for the chlorophyll a in peridinin-chlorophyll a-protein where the chlorophyll is shielded by protein. These results reflect varying degrees of protection of the tetrapyrrolic chromophores from the enzymatic oxidation and prove that bilirubin oxidase can be generally used as a probe for deducing the topography of tetrapyrrolic chromophores.
Singh
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BR
Department of Chemistry, School of Biological Sciences, University of Nebraska, Lincoln 68588.
Choi
J
J
Kwon
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TI
Song
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D000818:Animals; D002734:Chlorophyll / Q000378:metabolism*; D000077194:Chlorophyll A; D004141:Dinoflagellida / Q000378:metabolism*; D007700:Kinetics; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010798:Phycocyanin / Q000378:metabolism*; D010834:Phytochrome / Q000378:metabolism*; D010860:Pigments, Biological / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011758:Pyrroles / Q000378:metabolism*; D013053:Spectrophotometry / Q000379:methods; D045725:Tetrapyrroles
D010860:Pigments, Biological; D010940:Plant Proteins; D011506:Proteins; D011758:Pyrroles; D045725:Tetrapyrroles; D010798:Phycocyanin; D010834:Phytochrome; D002734:Chlorophyll; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C042401:bilirubin oxidase; D000077194:Chlorophyll A