The apoenzyme of NADPH oxidoreductase, 'old yellow enzyme', was reconstituted with specifically "N-labeled flavin mononucleotide and investigated by ' N NMR spectroscopy in the oxidized and reduced state.The results indicate (hat in (he oxidized state a hydrogen bond is formed between the N(5) atom and the apoprotein. In addition. hydrogen bonds exist between the N(l) and N(3) atoms of FMN and thc apoprotein. The resonance position ofN( 10) indicates that this atom is somewhat sp'-hybridized, i.e. lifted o u t ofthe molecular plane of thc isoalloxazine ring system.In the reduced state the N ( l ) atom is negatively charged and the N(3) atom forms a hydrogcn bond with the apoprotein. The N(10) atom in protein-bound FMN exhibits about the same hybridization state a s in free anionic reduced FMN, i.e. it is located in the plane of the isoalloxazine ring. The chemical shift of the N(5) resonance indicates that this atom is almost completely sp'-hybridized. This interpretation can also be derived from the 15N(5)-'H coupling constant. Among the flavoproteins thus fhr studied by NMR techniques, old yellow enzytne is the only protein that shows a conformation of the reduced prosthetic group with the N(5) atom lirted out of the molecular plane.'l'he isoelectric focussing properties of old yellow cnzyme and a new easy method for the preparation of the apoprotein are also reported.
The apoenzyme of NADPH oxidoreductase, 'old yellow enzyme', was reconstituted with selectively ' Cenriched flavin mononucleotides and investigated by " C NMR spectroscopy. The I3C N M R results confirm the results obtained by "N NMR spectroscopy and yield additional information about the coenzyme-apoenzyme interaction. A strong deshielding of the C(2) and C(4) atoms of enzyme-bound F M N both in the oxidized and reduced state is observed, which is supposed to be induced by hydrogen-bond formation between the protein and the two carbonyl groups at C(2) and C(4) of the isoalloxazine ring system. The chemical shifts of all 13C resonances of the flavin in the two-electron-reduced state indicate that the N(5) atom is sp3-hybridized.From ."P NMR measurements it is concluded that the F M N phosphate group is not accessible to bulk solvent. The unusual 'IP chemical shift of F M N in old yellow enzyme seems to indicate a different binding mode of the F M N phosphate group in this enzyme as compared to the flavodoxins.The I3C and "N N M R data on the old-yellow-enzyme-phenolate complexes show that the atoms of the phenolate are inore deshielded whereas the atoms of the enzyme-bound isoalloxazine ring are more shielded upon complexation. A non-linear correlation exists between the chemical shifts of the N(5) and the N(10) atoms and thepK, value of the phenolate derivative bound to the protein. Since the chemical shifts ofN(S), N(10) and C(4a) are influenced most on complexation it is suggested that the phenolate is bound near the pyrazine ring of the isoalloxazine system. "N N M R studies on the complex between FMN and 2-aminobenzoic acid indicate that the structure of this complex differs from that of the old-yellow-enzyme -phenolate complexes.There is considerable interest in the nature of the deeply coloured complexes, usually referred to as 'charge-transfer' complexes, formed by many flavoproteins by the interaction with certain organic compounds [I]. Although the catalytic function of old yellow enzyme is not known, it often serves as a model for physical studies of the coloured complexes. The suggestion that the phenolate binding of old yellow enzyme, which results in inhibition of the NADPH oxidase activity, could be related to the reaction with the physiological substrate [2-41 did not yet lead to the discovery of the catalytic function of the enzyme. We have untertaken an N M R study on this protein in the hope of contributing to a better understanding of the interaction between the apoprotein and the prosthetic group and the physical properties of the protein-phenolate complexes. There was some expectation that these investigations could also yield a valuable clue with respect to the catalytic function of the redox protein.
Die Messunsicherheit eines komplexen Analysenverfahrens lässt sich mit Softwareunterstützung einfach und schnell beziffern – wie die Bestimmung von Di‐n‐butylphthalat in Polyvinylchlorid zeigt.
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