On the Enigma of Old Yellow Enzyme's Spectral Properties

Abstract

Old yellow enzyme (NADPH oxidoreductase) in the free and complexed state was thoroughly investigated by the following techniques : absorption, circular dichroism, fluorescence/phosphorescence and electron paramagnetic resonance spectroscopy and fluorescence/phosphorescence decay measurements, applied over a wide range of temperature (7 -293 K). The data obtained were interpreted by comparison with results from similar measurements on free FMN, existing spectral data on isoalloxazine model systems and theoretical data.The results clearly demonstrate the inadequacy of a simple phenolate-FMN donor-acceptor charge-transfer complex to explain the phenomena occurring upon the addition of phenols to old yellow enzyme. Instead it was found that the phenolate anion interferes strongly with an existing tight complex between FMN and the apoprotein, probably an H-bonded structure in which FMN is tautomerized and interacts with an L-chiral center. This is concluded from a separate electronic transition with an origin at 496 nm, thus far not recognized as such, and the circular dichroism observed.The emission is dominated by that of free FMN, although protein-bound FMN seems also to become luminescent in glassy solution at 143 K. A second fluorescence/phosphorescence emission appears upon excitation of both native and complexed old yellow enzyme in the ultraviolet. This emission is quenched by the addition of phenol to the enzyme, shows a large (3000-cm-') blue shift on going to a low-temperature glass and is tentatively assigned to excimers of nucleic acids. Long-wavelength excitation with a synchronously pumped, mode-locked Rhodamine 6-G dye laser revealed a third, extremely weak emission in both native old yellow enzyme and its complexes. It decays with a lifetime of about 3 ns at 143 K. Electron paramagnetic resonance spectra revealed the presence of a low amount of an unpaired spin in old yellow enzyme. Owing to an unusual relaxational behaviour it could only be observed below 15 K and, again, the signal was measured in both the native enzyme and its complexes. Possible assignment and consequences of this observation are discussed.In frozen aqueous solutions of the enzyme-phenolate complex, a phase transition was discovered at which the colour of the complex reverted to that of the native enzyme. Subsequent melting restored the original colour.The observed phenomena and existing literature data lead to the conclusion that the only model from which no apparent inconsistencies emerge is that of a very complicated network of hydrogen-bonded structures in the protein. These involve several, partly unknown, chromophores. Phenols interfere with this network, leading to the formation of the long-wavelength absorption band in old yellow enzyme.The spectral changes induced by the binding of a lowmolecular-weight compound to old yellow enzyme (NADPH oxidoreductase) were initially regarded to be sufficiently mysterious to refer to this unknown compound as 'regreening factor' [I]. After the identification of this 'regreening factor'...

4-thioflavins as active site probes of flavoproteins: reaction with sulphite and formation of 4-hydroxy-4-sulphonylflavins

“…The 4-thio-FMN enzyme, like the native enzyme, also forms highly colored complexes with a variety of phenols. The absorption spectra of these complexes have been ascribed to charge transfer transitions between the bound phenolate anions and the oxidized flavin (Abramovitz and Massey, 1976b); this ascription has recently been questioned by Eweg et al (1982). The results obtained with enzyme reconstituted with 4-thio-FMN and derivatives which can be easily prepared from it (see Table IV) fully support our original conclusions.…”

Old yellow enzyme at 2 Å resolution: overall structure, ligand binding, and comparison with related flavoproteins

“…It had previously been proposed that the 'r electrons of the phenolic ring were interacting with the fr system of the flavin ring [3,38], and indeed, the two rings are largely parallel to each other, with the phenolate oxygen approaching within 3.0 A of flavin atom C2, and PHB atom C1 nearly 3.4 A above flavin N5. The hypothesis of Eweg et al [5], that the spectral changes were produced by the disruption of a hydrogen-bonding network that included other chromophores in addition to the flavin, is not borne out by the crystallographic data. Not only were no other chromophores detected, but binding of PHB does not appear to disrupt any interactions between the protein and the flavin.…”

“…The search has been heavily influenced by the observation that many phenolic compounds bind tightly to OYE giving rise to strong absorption maxima between 500 nm and 800 nm [4]. The spectral changes have been attributed to a charge-transfer complex between the ligand and the isoalloxazine ring of the flavin [3], although other explanations have been suggested [5]. Many phenolic compounds have been tested for activity, but none are turned over by the enzyme.…”

NAD(P)H‐Independent Asymmetric CC Bond Reduction Catalyzed by Ene Reductases by Using Artificial Co‐substrates as the Hydrogen Donor

“…Clearly, the five-membered hydroxyheteroaromatics formed after hydrogen abstraction from 46 c and 48 c result in less favorable p interactions with FMN than phenols or hydroquinones. [24,34,[36][37][38] 4) Not surprisingly, all co-substrates of type IV, lacking an electron-withdrawing group attached to the alkene moiety (51 c, 52 c), were inactive. Only compound 50 c, bearing an activating group in the exo-position, gave a moderate conversion.…”