Riboflavin is a component of the Na+-pumping NADH-quinone oxidoreductase from Vibrio cholerae

Abstract

Flavins are cofactors in many electron-transfer enzymes. Typically, two types of flavins perform this role: 5'-phosphoriboflavin (FMN) and flavin-adenine dinucleotide (FAD). Both of these are riboflavin derivatives, but riboflavin itself has never been reported to be an enzyme-bound component. We now report that tightly bound riboflavin is a component of the NADH-driven sodium pump from Vibrio cholerae.

Fig 1.

Chemical structures of riboflavin, FMN, and FAD. The covalently bound FMNs are attached to the protein via ester bonds between their phosphate groups (marked * in the figure) and threonine residues in subunits B and C.

Fig 2.

HPLC separation (a) and mass spectrometric (b) analysis of the soluble flavin components extracted from Na⁺-NQR after denaturation by 6 M guanidine-HCl. Shown is an HPLC elution profile of the extracted flavins monitored by the absorbance at 450 nm. The mass spectra of the two HPLC fractions are shown, corresponding to the expected masses of FAD and riboflavin. (a, Inset) UV-visible spectra of the material in each of the major HPLC peaks.

Fig 3.

HPLC elution profiles of low molecular weight fractions from denatured Na⁺-NQR and from bacterial membrane preparations including internal standards and controls. (a) HPLC of flavins released from heat-denatured (boiled) Na⁺-NQR with and without added FAD as an internal standard. Solid line, 10 nmol of flavins extracted from boiled Na⁺-NQR; dashed line, same but with 5 nmol of FAD added before boiling the sample. There is a slight shift in the elution positions of the peaks that was within the range of variability observed between different samples. The FAD is not degraded, and the area of the riboflavin peak is not influenced by the inclusion of FAD to the sample before boiling. (b) Same as described for a but 5 nmol of FMN were added before boiling in place of FAD as internal standard. The FMN is not degraded, and the amounts of FAD and riboflavin are unchanged. (c) HPLC elution profiles of the soluble components from V. cholerae membrane preparations after cold-TCA denaturation showing the presence of riboflavin in membranes from wild-type (WT) cells and its absence in a strain where the genes encoding Na⁺-NQR have been deleted. Riboflavin was used as both an internal and external standard as indicated in the labels.