The Binding of Flavins by Apoflavodoxins from Peptostreptococcus elsdenii and Azotobacter vinelandii as Studied by Temperature‐Jump Technique

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Abstract
The binding of various flavins by apoflavodoxins from P. elsdenii and A. vinelandii has been studied by the temperature‐jump technique using fluorescence detection. P. elsdenii apoflavodoxin interacts only with flavins possessing 5 carbon atoms in the N(10) side chain and a terminal phosphate group. Employing a wide range of concentrations of deoxy‐FMN Flavin = 3,4‐dimethyl‐lO‐substituted isoalloxazine = 3,4‐dimethyl‐ lO‐substituted‐2,3,4,10‐tetrahydro‐benzo[g]‐pteridine‐2,4‐dione; FMN = riboflavin‐5′‐monophosphate. and apoflavodoxin only one relaxation process was observed, indicating a one‐step binding mechanism. With native flavodoxin no relaxation could be observed.The kinetic parameters of the interaction of A. vinelandii apoflavodoxin with various flavin analogs (Structure I) have also been investigated. The interaction between apoflavodoxin and flavin derivatives carrying an ionizable, terminal functional group on the side chain becomes very weak when the number of the side chain carbon atoms is decreased below 4. This observation is interpreted in terms of repulsive forces due to negatively charged amino acid residues located in the flavin side chain binding region of the apoflavodoxin. All complexes studied revealed only one relaxation process. This observation is in contradiction with published results [10]. The published traces are instrumental artifacts.