Use of riboflavin-binding protein to investigate steric and electronic relationships in flavin analogs and models.

Abstract

We have examined the affinity of two recently synthesized flavin analogs for the isoalloxazine binding site of riboflavin-binding protein (RBP). The results showed that pyrimidopteridines could bind to . This suggested that, at the FMN or FAD level, these analogs might also bind to other apoflavoproteins, thereby providing a high potential probe for flavin enzymology. In contrast, 4a,5-ringopened isoalloxazines did not bind to RBP. However, 1,10a-ring-opened flavins bind with considerable avidity (Kd about 40 nM). Evidence is presented which indicates that the 4a,5-ring-opened species adopted a nonplanar configuration which, in turn, was responsible for the lack of affinity to RBP. Steric and electronic consequences of a 4a,5 ring opening are discussed in relation to flavin-dependent phenolic hydroxylases.Riboflavin-binding protein (RBP) is a protein of small molecular weight, whose primary physiological function is storage of vitamin B2 (riboflavin, formula I in Scheme 1) (1). Similar to other flavoproteins (2), its "active site" is able to bind not only the native "cofactor" but also various analogs in which the isoalloxazine nucleus of riboflavin has been modified (3-8). In contrast to other flavoproteins, in which the N(10) side chain of the cofactor (ribityl phosphate in case of FMN, ribityl diphosphate adenosyl for FAD) has to be highly preserved to accomplish binding and catalytic function, RBP tolerates modifications of the ribityl side chain. It therefore binds a variety of molecules that resemble flavins in their steric and electronic characteristics (3,4,6,9). Due to that feature, the isoalloxazine binding site of RBP has been well characterized by physical methods and a variety of probes (10-13). It is considered to be hydrophobic, and a tyrosine and a tryptophan are thought to be part of it. Their interaction with flavins and other probes is considered to be responsible for the fluorescence quenching of bound molecules. "Sandwiching" the flavin between them may account for the preferential binding of molecules with flat geometries. Weak binding of negatively charged molecules is ascribed to the presence of a negative charge in the flavin-binding site of the protein.Recently, two additional types of flavin models have become available for investigation (Scheme 1). The 4a,5-ringopened isoalloxazines (6-{[2-(dimethylamino)-4,5-dimethylphenyl]methylamino}-5-oxo-3H,5H-uracils) Ha (R3 = CH3) and lIb (R3 = H) were synthesized (14-16) to compare their spectral properties to those of enzyme-bound intermediates, observed during the hydroxylation reaction of flavindependent phenolic hydroxylases (17). Pyrimidopteridines (2,3,4,6,7,8,9 -heptahydro -2,4,6,8 -tetraoxopyrimido -5, -4g-pteridines), III (R3 or R7 = H or CH3), were designed to provide flavin cofactor analogs with more negative redox potentials than the native FMN of FAD (18). This would enable us to gain mechanistic insights by studying the reverse reactions of flavoenzymes with high redox potentials.The UV/visible spectra of the compounds II...

Catalytic Mechanism of 2-Hydroxybiphenyl 3-Monooxygenase, a Flavoprotein from Pseudomonas azelaica HBP1

“…However, in analogy with p-hydroxybenzoate hydroxylase (11,13,27) and phenol hydroxylase (19,34), the decay of the C(4a)-hydroxyflavin to oxidized enzyme was considerably retarded. The decay of intermediate III was not affected by binding of excess substrate as it was observed for phenol hydroxylase (34).…”

“…This intermediate is seen only with p-hydroxybenzoate hydroxylase (11,13) and phenol hydroxylase (18,19,25) in the presence of certain substrate analogs. Several models for the structure of intermediate II have been postulated (11,26,27), but all the evidence presently available suggests that intermediate II is a complex between the flavin C(4a)-hydroxide and the quinoid form of the aromatic product (19,24). The flavin C(4a)-hydroxide (intermediate III) is formed during the catalytic cycle after the oxygen transfer onto the substrate has occurred (11).…”

Altered Balance of Half-reactions in p-Hydroxybenzoate Hydroxylase Caused by Substituting the 2′-Carbon of FAD with Fluorine

“…The most extensively studied alternate substrate is 2,4-diOHB (11,26). An additional intermediate with a high extinction coefficient, called "intermediate II," forms upon transferring the distal oxygen atom of the flavin hydroperoxide to the substrate.…”

Biochemical Fluorometric Method for the Determination of Riboflavin in Milk

“…To date, three facts are firmly established: the exceptionally high association constant of RBF to RBP in water solutions, pH 6-9, on the order of 7.7 × 10 8 M -1 , the one-to-one stoichiometry, and the complete quenching of the fluorescence of RBF when complexed to RBP (3). From the crystal structure of RBP (4) it emerges how the binding site can form tight complexes with RBF (5,6). Fluorescence quenching has been ascribed to the stacking of the isoalloxazine ring of RBF with the Trp 156 and Tyr 75 aromatic rings of RBP, strategically present in the site together with four additional tryptophans clustering in the vicinity of the site (6).…”