Fluorescence Lifetimes As A Monitor Of Conformational Distributions In Nos
FASEB JOURNAL(2012)
摘要
The effects of calmodulin activation on the fluorescence of nitric oxide synthases are well established and parallel the activation of the enzyme by stimulation of electron transfer from NADPH to heme via the flavin containing domains. During this process, the FMN binding domain undergoes obligatory large scale conformational movements, resulting in the production of states in which the fluorescence lifetime of FMN, typically ~4.5 ns, is modified by interactions with FAD or heme, producing shorter lived states with lifetimes of ~90 ps or 1 ns. Changes in fluorescence have been observed by previous workers in response to calmodulin binding, but salt and chaotrope effects have also been observed. The enzyme has a pronounced dependence on ionic strength, characteristic of shuttle mechanisms, and some activities are greatly enhanced by chaotropes. Ionic strength has a relatively small effect on the distribution of lifetime states in the absence of calmodulin, consistent with the inability of salt to initiate NO synthesis or greatly enhance cytochrome c reduction in eNOS or nNOS. Chaotropes including guanidinium and urea, on the other hand, are effective at promoting the long lifetime states assigned to the free FMN domain (~4.3 ns) and the FMN‐oxygenase complex (1ns) at the expense of the short lifetime ‘input’ state in which FMN is in close association with FAD. The temperature dependence of the fluorescence decay indicates that increasing the temperature from 4C to 37C progressively favors the input state, in which the FMN binding domain has a large surface area of interaction with the FAD and NADPH binding domains. This suggests an entropy driven association in which reduction in size of a shell of ordered water provides the free energy.
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关键词
fluorescence,conformational distributions,nos
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