Ligand binding by the chlorocruorin from Eudistylia vancouverii.
Carbon monoxide chlorocruorin from Eudistylia vancouverii shows three distinct first-order relaxations with rates of 2.9 x 10(9) s-1, 6.5 x 10(7) s-1, and 3.2 x 10(6) s-1 (geminate reactions) and three second-order relaxations with rates of 4.7 x 10(6) M-1 s-1, 7 x 10(5) M-1 s-1, and 7 x 10(4) M-1 s-1, when studied by flash photolysis. The amplitudes of the second-order reactions depend on the extent of photolysis. This may be due to relaxation from the liganded (R) to the unliganded (T) conformation following photolysis and suggests that the combination rates contribute to cooperativity. In a stopped-flow experiment only the slowest phase with a rate of 7 x 10(4) M-1 s-1 is observed. It is assigned to binding to the T-state protein. Fragments of the native protein containing 12 and 4 hemes react like the holoprotein suggesting that the tetramer is a major cooperative unit. Oxygen binding shows three geminate relaxations with rates of 2.5 x 10(10) s-1, 3.5 x 10(7) s-1, and 4.5 x 10(6) s-1, and two second-order rates of 1.5 x 10(7) M-1 s-1 and 1 x 10(6) M-1 s-1. The amplitudes of the second-order phases do not correlate with the extent of photolysis. The results with the two ligands are consistent with an allosteric transition fast enough to compete with a rebinding rate of 500 s-1 in the R to T direction (CO rebinding) but not fast enough to compete with oxygen rebinding. There is significant heterogeneity in the R-state kinetics, but the T-state reaction is homogeneous.
Gibson
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QH
Department of Biochemistry and Molecular and Cellular Biology, Cornell University, Ithaca, New York 14853.
Bellelli
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A
Regan
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R
Sharma
P K
PK
Vinogradov
S N
SN
D000818:Animals; D001665:Binding Sites; D002248:Carbon Monoxide / Q000737:chemistry / Q000378:metabolism; D006418:Heme / Q000737:chemistry; D006420:Hemeproteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D010100:Oxygen / Q000378:metabolism; D010782:Photolysis; D011077:Polychaeta / Q000378:metabolism
D006420:Hemeproteins; D008024:Ligands; C010884:chlorocruorin; D006418:Heme; D002248:Carbon Monoxide; D010100:Oxygen