Old Yellow Enzyme (OYE) binds phenolic ligands forming long wavelength (500 -800 nm) charge-transfer complexes. The enzyme is reduced by NADPH, and oxygen, quinones, and ␣,␤-unsaturated aldehydes and ketones can act as electron acceptors to complete catalytic turnover. Solution of the crystal structure of OYE1 from brewer's bottom yeast (Fox, K. M., and Karplus, P. A. (1994) Structure 2, 1089 -1105) made it possible to identify histidine 191 and asparagine 194 as amino acid residues that hydrogen-bond with the phenolic ligands, stabilizing the anionic form involved in charge-transfer interaction with the FMN prosthetic group. His-191 and Asn-194 are also predicted to interact with the nicotinamide ring of NADPH in the active site. Mutations of His-191 to Asn, Asn-194 to His, and a double mutation, H191N/N194H, were made of OYE1. It was not possible to isolate the N191H mutant enzyme, but the other two mutant forms had the expected effect on phenolic ligand binding, i.e. decreased binding affinity and decreased charge-transfer absorbance. Reduction of the H191N mutant enzyme by NADPH was similar to that of OYE1, but the reduction rate constant for NADH was greatly decreased. The double mutant enzyme had an increased rate constant for reduction by NADPH, but the reduction rate constant with NADH was lower by a factor of 15. The reactivity of OYE1 and the mutant enzymes with oxygen was similar, but the reactivity of 2-cyclohexenone was greatly decreased by the mutations. The crystal structures of the two mutant forms showed only minor changes from that of the wild type enzyme. Old Yellow Enzyme (OYE;1 EC 1.6.99.1) is an NADPH oxidoreductase that contains flavin mononucleotide (FMN) as the prosthetic group. OYE was initially isolated from brewer's bottom yeast (1, 2) and was the first enzyme in which a vitaminderived molecule responsible for catalysis was shown to be associated with the protein in a 1:1 stoichiometry (3, 4). The protein was able to oxidize NADPH and, somewhat less efficiently, NADH (5, 6). The physiological oxidant of OYE has yet to be determined, but oxygen and a number of quinones can act in this capacity to complete the redox cycle (7). More recently it was found that 2-cyclohexenone (8) and a large number of other ␣,␤-unsaturated aldehydes and ketones are able to act as effective electron acceptors (9), suggesting that the physiological function of the enzyme might be the reduction of such a compound.Phenolic ligands bind to Old Yellow Enzyme with perturbation of the flavoprotein spectra and formation of striking long wavelength (500 -800 nm) absorbance bands (6, 10). This binding is affected by substituents on the phenol and a correlation between the energy of the charge-transfer absorption, and the Hammett para constant has been demonstrated. This correlation and an associated one with the redox potential of the flavin (11) have been used as evidence that the phenolate ion is the charge-transfer donor in the enzyme-phenol complex and the isoalloxazine ring of FMN is the acceptor (5, 12).OYE is...

In 1993, the first gene of Old Yellow Enzyme (OYE) of Saccharomyces cerevisiae was cloned (Stott, K., Saito, K., Thiele, D. J., and Massey, V. (1993) J. Biol. Chem. 268, 6097-6106) and named OYE2 to distinguish it from the first OYE gene cloned from Saccharomyces carlsbergenesis (Saito, K., Thiele, D. J., Davio, M., Lockridge, O., and Massey, V. (1991) J. Biol. Chem. 266, 20720-20724). The analysis of an OYE2 deletion mutant suggested that S. cerevisiae had at least two OYE genes. In the present study, we cloned a new OYE species named OYE3 and analyzed the OYE3 protein expressed in Escherichia coli. OYE3 consists of 400 amino acid residues and its molecular mass calculated by electrospray mass spectrometry is 44,788 daltons, in good agreement with the value of 44,920 daltons predicted from the amino acid sequence derived from the DNA sequence. In the downstream region of the OYE3 gene, the cytochrome oxidase (COX10) gene exists with a 426-base pair intermediate sequence. Some of the physicochemical and kinetic properties of OYE2 and OYE3 have been determined. Although the two enzymes are clearly closely related, they show differences in ligand binding properties and in their catalytic activities with oxygen and cyclohexen-2-one as acceptors.

Glutamine 114 of OYE1 is a well conserved residue in the active site of the Old Yellow Enzyme family. It forms hydrogen bonds to the O2 and N3 of the flavoprotein prosthetic group, FMN. Glutamine 114 was mutated to asparagine, introducing an R-group that is one methylene group shorter. The resultant enzyme was characterized to determine the effect of the mutation on the mechanistic behavior of the enzyme, and the crystal structure was solved to determine the effect of the mutation on the structure of the protein. The Q114N mutation results in little change in the protein structure, moving the amide group of residue 114 out of H-bonding distance, allowing repositioning of the FMN prosthetic group to form new interactions that replace the lost H-bonds. The mutation decreases the ability to bind ligands, as all dissociation constants for substituted phenols are larger than for the wild type enzyme. The rate constant for the reductive half-reaction with ␤-NADPH is slightly greater, whereas that for the oxidative half-reaction with 2-cyclohexenone is smaller than for the wild type enzyme. Oxidation with molecular oxygen is biphasic and involves formation and reaction with O 2 . , a phenomenon that is more pronounced with this mutation than with wild type enzyme. When superoxide dismutase is added to the reaction, we observe a single-phase reaction typical of the wild type enzyme. Turnover reactions using ␤-NADPH with 2-cyclohexenone and molecular oxygen were studied to further characterize the mutant enzyme.

The reaction of the old yellow enzyme and reduced flavins with organic nitrate esters has been studied. Reduced flavins have been found to react readily with glycerin trinitrate (GTN ) (nitroglycerin) and propylene dinitrate, with rate constants at pH 7.0, 25°C of 145 M ؊1 s ؊1 and 5.8 M ؊1 s ؊1 , respectively. With GTN, the secondary nitrate was removed reductively 6 times faster than the primary nitrate, with liberation of nitrite. With propylene dinitrate, on the other hand, the primary nitrate residue was 3 times more reactive than the secondary residue. In the old yellow enzyme-catalyzed NADPH-dependent reduction of GTN and propylene dinitrate, ping-pong kinetics are displayed, as found for all other substrates of the enzyme. Rapid-reaction studies of mixing reduced enzyme with the nitrate esters show that a reduced enzyme-substrate complex is formed before oxidation of the reduced flavin. The rate constants for these reactions and the apparent Kd values of the enzyme-substrate complexes have been determined and reveal that the rate-limiting step in catalysis is reduction of the enzyme by NADPH. Analysis of the products reveal that with the enzymecatalyzed reactions, reduction of the primary nitrate in both GTN and propylene dinitrate is favored by comparison with the freeflavin reactions. This preferential positional reactivity can be rationalized by modeling of the substrates into the known crystal structure of the enzyme. In contrast to the facile reaction of free reduced flavins with GTN, reduced 5-deazaflavins have been found to react some 4 -5 orders of magnitude slower. This finding implies that the chemical mechanism of the reaction is one involving radical transfers.

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