Kinetics and equilibria of active site core extrusion from spinach ferredoxin in aqueous N,N-dimethylformamide/Triton X-100 solutions.
The nature and reactivity of each species participating in core extrusion of the [2Fe-2S] cluster of spinach ferredoxin by benzenethiol have been investigated in a novel aqueous medium containing 10-40% (v/v) N,N-dimethylformamide (DMF) plus 5-6% (v/v) of the nonionic detergent, Triton X-100. By use of visible absorption and circular dichroism spectroscopies, it is found that prior to addition of benzenethiol, modifications of the ferredoxin induced by DMF are reversible. Both the extent and rate of modification are dependent on DMF concentration and on ionic strength. At ferredoxin concentrations near 0.1 mM, complete modification by DMF is shown to be unnecessary for quantitative core extrusion provided that the benzenethiol concentration is greater than or equal to 50 mM and the DMF concentration is greater than or equal to 10% (v/v). When benzenethiol is added after DMF, core extrusion occurs in two phases. Our interpretation is that the rapid phase corresponds to core extrusion of that portion of the ferredoxin already modified by DMF and that the slower phase corresponds to modification of the remaining portion of the ferredoxin by DMF. When DMF solutions containing benzenethiol and Triton X-100 in various ratios are mixed with aqueous solutions of spinach ferredoxin, the rate of core extrusion appears to be determined both by the rate at which the ferredoxin is modified and by the Triton/benzenethiol ratio. Under all conditions examined we observe significantly faster rates of core extrusion from spinach ferredoxin in aqueous Triton/DMF than in 80/20 (v/v) hexamethylphosphoramide/water, a previously used core extrusion medium. Our results suggest a catalytic role for the micellar phase.[1]References
- Kinetics and equilibria of active site core extrusion from spinach ferredoxin in aqueous N,N-dimethylformamide/Triton X-100 solutions. Bonomi, F., Kurtz, D.M. Biochemistry (1982) [Pubmed]
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