Dissecting the Energetics of the Apoflavodoxin-FMN Complex

Abstract

Many flavoproteins are non-covalent complexes between FMN and an apoprotein. To understand better the stability of flavoproteins, we have studied the energetics of the complex between FMN and the apoflavodoxin from Anabaena PCC 7119 by a combination of site-directed mutagenesis, titration calorimetry, equilibrium binding constant determinations, and x-ray crystallography. Comparison of the strength of the wild type and mutant apoflavodoxin-FMN complexes and that of the complexes between wild type apoflavodoxin and shortened FMN analogues (riboflavin and lumiflavin) allows the dissection of the binding energy into contributions associated with the different parts of the FMN molecule. The estimated contribution of the phosphate is greatest, at 7 kcal mol ؊1 ; that of the isoalloxazine is of around 5-6 kcal mol ؊1 (mainly due to interaction with Trp-57 and Tyr-94 in the apoprotein) and the ribityl contributes least: around 1 kcal mol ؊1 . The stabilization of the complex is both enthalpic and entropic although the enthalpy contribution is dominant. Both the phosphate and the isoalloxazine significantly contribute to the enthalpy of binding. The ionic strength does not have a large effect on the stability of the FMN complex because, although it weakens the phosphate interactions, it strengthens the isoalloxazine-protein hydrophobic interactions. Phosphate up to 100 mM does not affect the strength of the riboflavin complex, which suggests the isoalloxazine and phosphate binding sites may be independent in terms of binding energy. Interestingly, we find crystallographic evidence of flexibility in one of the loops (57-62) involved in isoalloxazine binding.Many proteins use tightly bound cofactors to perform their biological functions. Flavoproteins, redox proteins carrying a flavin group, are one example (1). Although there are several flavoproteins where the flavin is covalently bound (2), the large majority consist on tight, but non-covalent, complexes of apoprotein and flavin. The flavins in flavoproteins derive from riboflavin and come in two lengths. The short one, flavin mononucleotide (FMN; Fig. 1) is just 5Ј phosphoriboflavin; the long one is flavin adenine dinucleotide (FAD). In either flavin, the redox properties are confined to the isoalloxazine ring, the rest of the molecule being important for binding. FMN and FAD are not usually interchangeable. On binding to the protein, the redox properties of the flavin are tailored to suit the particular requirements of the redox reaction where the protein is involved (3-5).One of the smallest and better known flavoproteins is flavodoxin. Flavodoxin is an ␣/␤ electron transfer protein, involved in both photosynthetic and non-photosynthetic reactions, that carries a molecule of non-covalently bound FMN as its only redox center (6, 7). The FMN can be removed from the protein, and the resulting apoflavodoxin can be easily reconstituted. Although there are several studies on the interaction of FMN with apoflavodoxins (4, 6, 8 -10), a detailed analysis of the energetics of...

Molecular determinants for FMN‐binding in Desulfovibrio gigas flavoredoxin

“…The effects on K d of the double mutation, T56A, K92A, and of the triple mutation, N29A, T56A, K92A, indicate that the interactions between these residues and the FMN phosphate are synergistic. A similar synergistic behavior in the binding of the phosphate of FMN was previously reported for flavodoxin from Anabaena PCC7119 [18]. H131 in D. gigas Flr occurs in a triplet motif (GDH) that is characteristic of certain flavin reductases, proposed to be a major determinant in the interaction of nicotinamide nucleotides with such enzymes and very close to the isoalloxazine structure of the flavin in the proposed structure of Flr.…”

Electrochemical switching of the flavoprotein dodecin at gold surfaces modified by flavin-DNA hybrid linkers

“…1, dodecin loads binding energy predominantly on the isoalloxazine substructure, keeping the aliphatic chain rather unrestricted. 7,8 The dodecin binding mode is reflected by dissociation constants ͑K d 's͒ for lumiflavin ͑17.6 nM͒, riboflavin ͑35.8 nM͒, and CofC6 ͑46.9 nM͒, which are almost independent of the N͑10͒ substituent at the isoalloxazine moiety.…”

The ribosome regulates flavodoxin folding

“…FMN is shown in yellow. The cartoon rendering of MG off is a model for the known secondary structure elements within the off-pathway MG. α-helices in MG off that are also found in native apoflavodoxin are coloured green, non-native helices in MG off are coloured blue and the structured part of MG off that is neither α-helix nor β-strand is coloured orange (82). This model does not represent the relative positioning of these secondary structure elements within MG off , because this positioning is currently unknown.…”

“…In flavodoxin variant F44Y, in which phenylalanine at position 44 is substituted for a tyrosine, MG off can be induced in the absence of denaturant by lowering the ionic strength (82). This mutation de facto introduces an oxygen atom into a hydrophobic pocket of apoflavodoxin and destabilizes its native state (78,82).…”

“…This mutation de facto introduces an oxygen atom into a hydrophobic pocket of apoflavodoxin and destabilizes its native state (78,82). F44Y apoflavodoxin is thus an interesting candidate to study formation of off-pathway MGs in vivo, as substantial population of MG off occurs at physiologically relevant conditions.…”