19F NMR Studies with 2′-F-2′-deoxyarabinoflavoproteins

Apoproteins of several flavoproteins were reconstituted with 2′-F-2′-deoxyarabinoflavins and studied by 19F NMR and absorption spectroscopy. Extensive protein-fluorine interactions were observed by large chemical shift changes on binding to the apoprotein of Old Yellow Enzyme (apoOYE) and apoflavodoxin, whereas binding to apoglucose oxidase and apo D-amino acid oxidase (apoDAAO) resulted in minimal interactions. Modification at the flavin 2′-position in OYE resulted in a substantial decrease in the binding affinity of the flavin, possibly from the disruption of two important hydrogen bonds to Pro-35 and Arg-243. 19F NMR studies of complexes of OYE with testosterone, cyclohexenone, and β-estradiol suggest that phenols and α,β-unsaturated ketones orient differently at the active site on binding. The two separate one-electron potentials for the EFlox/EFlsq and EFlsq/EFlred couples were different for the reconstituted OYE. With native enzyme, there is 15-20% thermodynamic stabilization of the anionic flavin semiquinone, while no detectable amount of semiquinone was observed with modified OYE. This change in potential was further substantiated by blue shifts for the maxima of the modified protein-phenol charge transfer complexes. In accordance with the crystal structure of the OYE-p-OH-benzaldehyde complex (Fox, K.M. & Karplus, P.A. (1994) Structure 2, 1089-1105), 19F NMR studies with the modified OYE-2,4-F2-phenol suggest strong interaction between the para-fluorine of the phenol and Tyr-375. Apoproteins of several flavoproteins were reconstituted with 2′-F-2′-deoxyarabinoflavins and studied by 19F NMR and absorption spectroscopy. Extensive protein-fluorine interactions were observed by large chemical shift changes on binding to the apoprotein of Old Yellow Enzyme (apoOYE) and apoflavodoxin, whereas binding to apoglucose oxidase and apo D-amino acid oxidase (apoDAAO) resulted in minimal interactions. Modification at the flavin 2′-position in OYE resulted in a substantial decrease in the binding affinity of the flavin, possibly from the disruption of two important hydrogen bonds to Pro-35 and Arg-243. 19F NMR studies of complexes of OYE with testosterone, cyclohexenone, and β-estradiol suggest that phenols and α,β-unsaturated ketones orient differently at the active site on binding. The two separate one-electron potentials for the EFlox/EFlsq and EFlsq/EFlred couples were different for the reconstituted OYE. With native enzyme, there is 15-20% thermodynamic stabilization of the anionic flavin semiquinone, while no detectable amount of semiquinone was observed with modified OYE. This change in potential was further substantiated by blue shifts for the maxima of the modified protein-phenol charge transfer complexes. In accordance with the crystal structure of the OYE-p-OH-benzaldehyde complex (Fox, K.M. & Karplus, P.A. (1994) Structure 2, 1089-1105), 19F NMR studies with the modified OYE-2,4-F2-phenol suggest strong interaction between the para-fluorine of the phenol and Tyr-375. INTRODUCTIONIn order to understand the various mechanisms of flavin reactions, knowledge of the protein environment at the active centers of different flavoenzymes is necessary. Several physical and chemical methods are available for this purpose. Of these, replacement of the native flavin with appropriately modified flavins and high resolution NMR techniques constitute very effective and widely exploited tools. The former method in general provides information about the solvent accessibility of the modified position, hydrophobicity, and steric constraints of the binding site (1Ghisla S. Massey V. Biochem. J. 1986; 239: 1-12Crossref PubMed Scopus (166) Google Scholar). In the latter technique, comparison of the chemical shifts of the protein-bound flavin with the free flavin as well as the oxidized and reduced flavoproteins provides direct information about alterations in the magnetic environment of the substituent fluorine and hence can provide information about the protein environment surrounding the flavin (2Beinert W. Ruterjans H. Muller F. Eur. J. Biochem. 1985; 152: 573-579Crossref PubMed Scopus (42) Google Scholar, 3Beinert W. Ruterjans H. Muller F. Bacher A. Eur. J. Biochem. 1985; 152: 581-587Crossref PubMed Google Scholar, 4Macheroux P. Kojiro C.L. Schopfer L.M. Chakraborty S. Massey V. Biochemistry. 1990; 29: 2670-2679Crossref PubMed Scopus (14) Google Scholar). Old Yellow Enzyme is the oldest of the flavoprotein family and, as isolated from brewer's bottom yeast, is a mixture of homodimers and a heterodimer arising from two separate yeast genes, with each monomeric unit containing one FMN (5Schopfer L.M. Massey V. Kuby S.A. A Study of Enzymes. CRC Press, Inc., Boca Raton, FL1990: 247-283Google Scholar, 6Stott K. Saito K. Thiele D.J. Massey V. J. Biol. Chem. 1993; 268: 6097-6106Abstract Full Text PDF PubMed Google Scholar). Due to the fact that the physiological role of this enzyme is yet to be determined, the structure and reactivity of this protein has been the subject of extensive studies in our laboratory. Recent x-ray crystallographic studies suggest an important role for the 2′-hydroxyl group of the FMN in binding to the apoprotein and also in forming the binding site for various ligands (7Fox K.M. Karplus P.A. Yagi K. Flavins and Flavoproteins 1993. Walter de Gruyter, Berlin1994: 381Crossref Google Scholar, 8Fox K.M. Karplus P.A. Structure. 1994; 2: 1089-1105Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). This hydroxyl group is involved in two important hydrogen bonds with Pro-35 and with Arg-243, one of the positively charged residues around the flavin N1 (Fig. 1). It was thought that the disruption of these hydrogen bonds would affect the configuration of the net charge at the binding site and so it was of obvious interest to study how this would affect the flavin binding to apoprotein, binding of various ligands, and catalytic activity. A new flavin, 2′-fluoro-2′-deoxyarabinoriboflavin was designed, where the 2′-hydroxyl group of the ribityl side chain was replaced with a hydrogen and a fluorine was incorporated in place of the hydrogen at the same 2′-carbon (9Murthy Y.V.S.N. Massey V. J. Biol. Chem. 1995; 270: 28586-28594Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 10Miller S.M. Biochemistry. 1995; 34: 13066-13073Crossref PubMed Scopus (7) Google Scholar). This flavin should be an ideal probe for ligand-binding studies and also provide information on the chemical environment of the flavin binding site in terms of 19F chemical shifts. It was also thought that this flavin could be a good 19F NMR probe for flavoproteins in general. The high sensitivity of 19F, which is next only to 1H, the wide range of chemical shifts, and the ease with which one can incorporate fluorine into biological molecules make 19F NMR a very effective physical tool to study proteins (11Sykes B.D. Hull W.E. Methods Enzymol. 1978; 49: 270-295Crossref PubMed Scopus (61) Google Scholar, 12Greig J.P. Berliner L.J. Rueben J. Biological Magnetic Resonance. Plenum, New York1978: 139Google Scholar, 13Sykes B.D. Weiner J.H. Cohen J.S. Magnetic Resonance in Biology. John Wiley and Sons, Inc., New York1980: 171Google Scholar). Another important feature is that the number of peaks for each fluorine correspond to the number of binding environments present in the protein. Since the fluorine is in the ribityl side chain of this flavin, the net electron environment (fluorine is highly electronegative) of the isoalloxazine ring of the flavin is unperturbed by its presence, unlike in the case of 8-F-flavins, where the benzene ring of the flavin experiences more positive character due to the presence of the electronegative fluorine (4Macheroux P. Kojiro C.L. Schopfer L.M. Chakraborty S. Massey V. Biochemistry. 1990; 29: 2670-2679Crossref PubMed Scopus (14) Google Scholar). Another advantage of fluorine being on the hydrophilic ribityl side chain is that it interacts more with the protein; and changes in the fluorine chemical shifts are expected to be very sensitive to capture even minute changes in the protein conformation. In the case of 8-F-flavin, fluorine being on the hydrophobic benzene ring of the flavin, the differences in chemical shifts are not that pronounced for most of the proteins studied (4Macheroux P. Kojiro C.L. Schopfer L.M. Chakraborty S. Massey V. Biochemistry. 1990; 29: 2670-2679Crossref PubMed Scopus (14) Google Scholar). INTRODUCTIONIn order to understand the various mechanisms of flavin reactions, knowledge of the protein environment at the active centers of different flavoenzymes is necessary. Several physical and chemical methods are available for this purpose. Of these, replacement of the native flavin with appropriately modified flavins and high resolution NMR techniques constitute very effective and widely exploited tools. The former method in general provides information about the solvent accessibility of the modified position, hydrophobicity, and steric constraints of the binding site (1Ghisla S. Massey V. Biochem. J. 1986; 239: 1-12Crossref PubMed Scopus (166) Google Scholar). In the latter technique, comparison of the chemical shifts of the protein-bound flavin with the free flavin as well as the oxidized and reduced flavoproteins provides direct information about alterations in the magnetic environment of the substituent fluorine and hence can provide information about the protein environment surrounding the flavin (2Beinert W. Ruterjans H. Muller F. Eur. J. Biochem. 1985; 152: 573-579Crossref PubMed Scopus (42) Google Scholar, 3Beinert W. Ruterjans H. Muller F. Bacher A. Eur. J. Biochem. 1985; 152: 581-587Crossref PubMed Google Scholar, 4Macheroux P. Kojiro C.L. Schopfer L.M. Chakraborty S. Massey V. Biochemistry. 1990; 29: 2670-2679Crossref PubMed Scopus (14) Google Scholar). Old Yellow Enzyme is the oldest of the flavoprotein family and, as isolated from brewer's bottom yeast, is a mixture of homodimers and a heterodimer arising from two separate yeast genes, with each monomeric unit containing one FMN (5Schopfer L.M. Massey V. Kuby S.A. A Study of Enzymes. CRC Press, Inc., Boca Raton, FL1990: 247-283Google Scholar, 6Stott K. Saito K. Thiele D.J. Massey V. J. Biol. Chem. 1993; 268: 6097-6106Abstract Full Text PDF PubMed Google Scholar). Due to the fact that the physiological role of this enzyme is yet to be determined, the structure and reactivity of this protein has been the subject of extensive studies in our laboratory. Recent x-ray crystallographic studies suggest an important role for the 2′-hydroxyl group of the FMN in binding to the apoprotein and also in forming the binding site for various ligands (7Fox K.M. Karplus P.A. Yagi K. Flavins and Flavoproteins 1993. Walter de Gruyter, Berlin1994: 381Crossref Google Scholar, 8Fox K.M. Karplus P.A. Structure. 1994; 2: 1089-1105Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). This hydroxyl group is involved in two important hydrogen bonds with Pro-35 and with Arg-243, one of the positively charged residues around the flavin N1 (Fig. 1). It was thought that the disruption of these hydrogen bonds would affect the configuration of the net charge at the binding site and so it was of obvious interest to study how this would affect the flavin binding to apoprotein, binding of various ligands, and catalytic activity. A new flavin, 2′-fluoro-2′-deoxyarabinoriboflavin was designed, where the 2′-hydroxyl group of the ribityl side chain was replaced with a hydrogen and a fluorine was incorporated in place of the hydrogen at the same 2′-carbon (9Murthy Y.V.S.N. Massey V. J. Biol. Chem. 1995; 270: 28586-28594Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 10Miller S.M. Biochemistry. 1995; 34: 13066-13073Crossref PubMed Scopus (7) Google Scholar). This flavin should be an ideal probe for ligand-binding studies and also provide information on the chemical environment of the flavin binding site in terms of 19F chemical shifts. It was also thought that this flavin could be a good 19F NMR probe for flavoproteins in general. The high sensitivity of 19F, which is next only to 1H, the wide range of chemical shifts, and the ease with which one can incorporate fluorine into biological molecules make 19F NMR a very effective physical tool to study proteins (11Sykes B.D. Hull W.E. Methods Enzymol. 1978; 49: 270-295Crossref PubMed Scopus (61) Google Scholar, 12Greig J.P. Berliner L.J. Rueben J. Biological Magnetic Resonance. Plenum, New York1978: 139Google Scholar, 13Sykes B.D. Weiner J.H. Cohen J.S. Magnetic Resonance in Biology. John Wiley and Sons, Inc., New York1980: 171Google Scholar). Another important feature is that the number of peaks for each fluorine correspond to the number of binding environments present in the protein. Since the fluorine is in the ribityl side chain of this flavin, the net electron environment (fluorine is highly electronegative) of the isoalloxazine ring of the flavin is unperturbed by its presence, unlike in the case of 8-F-flavins, where the benzene ring of the flavin experiences more positive character due to the presence of the electronegative fluorine (4Macheroux P. Kojiro C.L. Schopfer L.M. Chakraborty S. Massey V. Biochemistry. 1990; 29: 2670-2679Crossref PubMed Scopus (14) Google Scholar). Another advantage of fluorine being on the hydrophilic ribityl side chain is that it interacts more with the protein; and changes in the fluorine chemical shifts are expected to be very sensitive to capture even minute changes in the protein conformation. In the case of 8-F-flavin, fluorine being on the hydrophobic benzene ring of the flavin, the differences in chemical shifts are not that pronounced for most of the proteins studied (4Macheroux P. Kojiro C.L. Schopfer L.M. Chakraborty S. Massey V. Biochemistry. 1990; 29: 2670-2679Crossref PubMed Scopus (14) Google Scholar).