A 13C Nuclear‐Magnetic‐Resonance Study on Free Flavins and Megasphaera elsdenii and Azotobacter vinelandii Flavodoxin

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Abstract
Selectively 13C‐enriched free and protein‐bound flavins in the oxidized and two‐electron reduced state were investigated by the 13 C nuclear magnetic resonance technique. FMN in aqueous solution and N‐(3)‐menthyl‐2′,3′4,'5′‐tetraacetylriboflavin in an apolar solvent were used as reference compounds for the protein‐bound FMNA comparison of th chemical shifts of FMN in aqueous solution with those of N(3)‐methyltetraacetyl‐ riboglavin in CHCL3 reveals that FMN is strongly polarized yielding a pseudo‐ionic molecule stabilized by hydrogen bonding of H2O with C‐2α of the isoalloxazine molecule. The interpretation is fully supported by the one‐bond)1J) 13C coupling constants. The chemical shifts combined with the coupling constants indicate the change of charge distribution within the molecule when going from apolar to polar solutionsBinding of FMN to Megasphaera elsdenii and to Azotobacter vinelandii apoflavodoxins affects the resonances due to C‐2 and C‐10 a as compared to free FMN.These shifts together with the coupling constants indicate the formation of a hydrogen bound between C‐2α of FMN and an amino acid residue of the apoprotein. In M. Elsdenii flavodoxin such a hydrogen bond also exists with C‐4α which is not observed in A vinelandii flavodoxin.The chemical shifts to the two‐electron reduced derivatives of FMN and N(3)menthyl‐2′,3′,4′,5′,‐tetraacetyl‐ riboflavin clearly indicate that the FMN derivative possesses a more planar conformation than its analog; it shows pH‐independent, From the pH‐dependence of the chemical shifts of the atoms C‐10a, C‐2 and C‐4a. The chemical shift of C‐4 is pH‐independent. From the pH‐ dependence of the chemical shifts an ionization constant of 6.7 is calculated.The chemical shifts of protein‐bound FMN in the reduced state are very similar to those of the anionic, reduced FMN. The data show that in both flavodoxins studied the prosthetic group when reduced is bound in the anionic form an possesses approximately a coplanar confornation.The results are discussed with respect to the possible biological implications. It is suggested that specific interaction of the prosthetic group with the apoprotein, the ionization state and the degree of planarity of the reduced prosthetic group are factors determining the biological functions of flavodoxins and possibly the functions of flavoproteins in general.