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αArg-237 in Methylophilus methylotrophus (sp. W3A1) Electron-transferring Flavoprotein Affords ∼200-Millivolt Stabilization of the FAD Anionic Semiquinone and a Kinetic Block on Full Reduction to the Dihydroquinone

2001; Elsevier BV; Volume: 276; Issue: 23 Linguagem: Inglês

10.1074/jbc.m010853200

1083-351X

François Talfournier, Andrew W. Munro, Jaswir Basran, Michael J. Sutcliffe, Simon Daff, Stephen K. Chapman, Nigel S. Scrutton,

Photosynthetic Processes and Mechanisms

The midpoint reduction potentials of the FAD cofactor in wild-type Methylophilus methylotrophus (sp. W3A1) electron-transferring flavoprotein (ETF) and the αR237A mutant were determined by anaerobic redox titration. The FAD reduction potential of the oxidized-semiquinone couple in wild-type ETF (E′1) is +153 ± 2 mV, indicating exceptional stabilization of the flavin anionic semiquinone species. Conversion to the dihydroquinone is incomplete (E′2 < −250 mV), because of the presence of both kinetic and thermodynamic blocks on full reduction of the FAD. A structural model of ETF (Chohan, K. K., Scrutton, N. S., and Sutcliffe, M. J. (1998) Protein Pept. Lett. 5, 231–236) suggests that the guanidinium group of Arg-237, which is located over the si face of the flavin isoalloxazine ring, plays a key role in the exceptional stabilization of the anionic semiquinone in wild-type ETF. The major effect of exchanging αArg-237 for Ala in M. methylotrophus ETF is to engineer a remarkable ∼200-mV destabilization of the flavin anionic semiquinone (E′2 = −31 ± 2 mV, andE′1 = −43 ± 2 mV). In addition, reduction to the FAD dihydroquinone in αR237A ETF is relatively facile, indicating that the kinetic block seen in wild-type ETF is substantially removed in the αR237A ETF. Thus, kinetic (as well as thermodynamic) considerations are important in populating the redox forms of the protein-bound flavin. Additionally, we show that electron transfer from trimethylamine dehydrogenase to αR237A ETF is severely compromised, because of impaired assembly of the electron transfer complex. The midpoint reduction potentials of the FAD cofactor in wild-type Methylophilus methylotrophus (sp. W3A1) electron-transferring flavoprotein (ETF) and the αR237A mutant were determined by anaerobic redox titration. The FAD reduction potential of the oxidized-semiquinone couple in wild-type ETF (E′1) is +153 ± 2 mV, indicating exceptional stabilization of the flavin anionic semiquinone species. Conversion to the dihydroquinone is incomplete (E′2 < −250 mV), because of the presence of both kinetic and thermodynamic blocks on full reduction of the FAD. A structural model of ETF (Chohan, K. K., Scrutton, N. S., and Sutcliffe, M. J. (1998) Protein Pept. Lett. 5, 231–236) suggests that the guanidinium group of Arg-237, which is located over the si face of the flavin isoalloxazine ring, plays a key role in the exceptional stabilization of the anionic semiquinone in wild-type ETF. The major effect of exchanging αArg-237 for Ala in M. methylotrophus ETF is to engineer a remarkable ∼200-mV destabilization of the flavin anionic semiquinone (E′2 = −31 ± 2 mV, andE′1 = −43 ± 2 mV). In addition, reduction to the FAD dihydroquinone in αR237A ETF is relatively facile, indicating that the kinetic block seen in wild-type ETF is substantially removed in the αR237A ETF. Thus, kinetic (as well as thermodynamic) considerations are important in populating the redox forms of the protein-bound flavin. Additionally, we show that electron transfer from trimethylamine dehydrogenase to αR237A ETF is severely compromised, because of impaired assembly of the electron transfer complex. electron-transferring flavoprotein trimethylamine dehydrogenase Electron-transferring flavoproteins (ETFs)1 act as carriers of electrons in bacteria and mitochondria. They mediate electron transfer between degradative enzymes and membrane-bound electron acceptors (1Thorpe C. Muller F. Chemistry and Biochemistry of Flavoenzymes. II. CRC Press, Inc., Boca Raton, FL1991: 471-486Google Scholar). ETFs have been classified into two functional groups (2Weidenhaupt M. Rossi P. Beck C. Fischer H.M. Hennecke H. Arch. Microbiol. 1996; 165: 169-178PubMed Google Scholar). Housekeeping ETFs function in the oxidation of fatty acids and some amino acids and have been isolated from mammalian and bacterial sources (1Thorpe C. Muller F. Chemistry and Biochemistry of Flavoenzymes. II. CRC Press, Inc., Boca Raton, FL1991: 471-486Google Scholar, 3Husain M. Steenkamp D.J. J. Bacteriol. 1985; 163: 709-715Crossref PubMed Google Scholar). Specialized ETFs are restricted to prokaryotes and are synthesized under defined nutritional conditions. Specialized ETFs are involved in the oxidation of trimethylamine (4Steenkamp D.J. Gallup M. J. Biol. Chem. 1978; 253: 4086-4089Abstract Full Text PDF PubMed Google Scholar) and carnitine (5Eichler K. Buchet A. Bourgis F. Kleber H.P. Mandrand-Berthelot M.A. J. Basic Microbiol. 1995; 35: 217-227Crossref PubMed Scopus (38) Google Scholar, 6Tsai M.H. Saier Jr., M.H. Res. Microbiol. 1995; 146: 397-404Crossref PubMed Scopus (45) Google Scholar) and are also important in nitrogen fixation (7Earl C.D. Ronson C.W. Ausubel F.M. J. Bacteriol. 1987; 169: 1127-1136Crossref PubMed Scopus (94) Google Scholar). All ETFs possess one equivalent of non-covalently bound FAD per ETF heterodimer, except the ETF from Megasphaera elsdenii, which contains 2 equivalents of FAD per dimer (8Whitfield C.D. Mayhew S.G. J. Biol. Chem. 1974; 249: 2801-2810Abstract Full Text PDF PubMed Google Scholar). It has been shown that AMP (1 equivalent) is associated with the housekeeping ETFs from pigs (9Sato K. Nishina Y. Shiga K. J. Biochem. ( Tokyo ). 1993; 114: 215-222Crossref PubMed Scopus (41) Google Scholar), humans (10Roberts D.L. Frerman F.E. Kim J.-J.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14355-14360Crossref PubMed Scopus (155) Google Scholar), and Paracoccus denitrificans (11Roberts D.L. Salazar D. Fulmer J.P. Frerman F.E. Kim J.J. Biochemistry. 1999; 38: 1977-1989Crossref PubMed Scopus (58) Google Scholar) and with the specialized ETF from Methylophilus methylotrophus(12Duplessis E.R. Rohlfs R.J. Hille R. Thorpe C. Biochem. Mol. Biol. Int. 1994; 32: 195-199PubMed Google Scholar). Mammalian and bacterial ETF proteins act as one-electron carriers, cycling between the oxidized and anionic flavin semiquinone forms. The ETF from M. elsdenii is unusual in acting physiologically as a two-electron carrier. ETF from mammalian sources and P. denitrificans can be reduced to the dihydroquinone form, by reduction with dithionite or by photoreduction (13Gorelick R.J. Mizzer J.P. Thorpe C. Biochemistry. 1982; 21: 6936-6942Crossref PubMed Scopus (72) Google Scholar, 14Husain M. Steenkamp D.J. Biochem. J. 1983; 209: 541-545Crossref PubMed Scopus (65) Google Scholar, 15Watmough N.J. Kiss J. Frerman F.E. Eur. J. Biochem. 1992; 205: 1089-1097Crossref PubMed Scopus (17) Google Scholar), although reduction to the two-electron level is relatively slow.M. methylotrophus ETF is readily converted to the semiquinone form in reactions with its physiological electron donor, trimethylamine dehydrogenase (TMADH) (4Steenkamp D.J. Gallup M. J. Biol. Chem. 1978; 253: 4086-4089Abstract Full Text PDF PubMed Google Scholar), or during artificial reduction with dithionite (16Davidson V.L. Husain M. Neher J.W. J. Bacteriol. 1986; 166: 812-817Crossref PubMed Google Scholar). However, further reduction to the dihydroquinone is not observed with dithionite (16Davidson V.L. Husain M. Neher J.W. J. Bacteriol. 1986; 166: 812-817Crossref PubMed Google Scholar) or with catalytic amounts of TMADH (4Steenkamp D.J. Gallup M. J. Biol. Chem. 1978; 253: 4086-4089Abstract Full Text PDF PubMed Google Scholar). Reduction of M. methylotrophus ETF to the dihydroquinone form can be achieved (albeit sluggishly) by electrochemical methods (17Byron C.M. Stankovich M.T. Husain M. Davidson V.L. Biochemistry. 1989; 28: 8582-8587Crossref PubMed Scopus (37) Google Scholar). In addition, when ETF is in complex with TMADH, the FAD is more readily reduced to the two-electron level (18Jang M.-H. Scrutton N.S. Hille R. J. Biol. Chem. 1999; 275: 12546-12552Abstract Full Text Full Text PDF Scopus (18) Google Scholar). In this latter case, further reduction to the dihydroquinone is likely to be a consequence of a large scale structural reorganization in ETF that accompanies complex assembly with TMADH (18Jang M.-H. Scrutton N.S. Hille R. J. Biol. Chem. 1999; 275: 12546-12552Abstract Full Text Full Text PDF Scopus (18) Google Scholar, 19Chohan K.K. Scrutton N.S. Sutcliffe M.J. Protein Pept. Lett. 1998; 5: 231-236Google Scholar, 20Jones M. Basran J. Sutcliffe M.J. Gunter Grossmann J. Scrutton N.S. J. Biol. Chem. 2000; 275: 21349-21354Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). The midpoint reduction potentials of the E′1(quinone-semiquinone) and E′2(semiquinone-dihydroquinone) couples of the FAD in M. methylotrophus ETF have been determined. The potential of the quinone-semiquinone couple is exceptionally high (+196 mV (17Byron C.M. Stankovich M.T. Husain M. Davidson V.L. Biochemistry. 1989; 28: 8582-8587Crossref PubMed Scopus (37) Google Scholar) and +141 mV (21Wilson E.K. Huang L. Sutcliffe M.J. Mathews F.S. Hille R. Scrutton N.S. Biochemistry. 1997; 36: 41-48Crossref PubMed Scopus (38) Google Scholar) as determined by electrochemical and spectrophotometric methods, respectively), consistent with a need to accept electrons from the 4Fe-4S center of TMADH (midpoint potential, +102 mV (22Barber M.J. Pollock V. Spence J.T. Biochem. J. 1988; 256: 657-659Crossref PubMed Scopus (25) Google Scholar)). The potential of the semiquinone-dihydroquinone couple is more conventional (-197 mV; (17Byron C.M. Stankovich M.T. Husain M. Davidson V.L. Biochemistry. 1989; 28: 8582-8587Crossref PubMed Scopus (37) Google Scholar)), indicating that there is a substantial (and essentially complete) kinetic block on full reduction of ETF by dithionite (-530 mV) or photoexcited deazariboflavin (-650 mV). A similar, albeit less complete, kinetic block on reduction to the dihydroquinone has been reported for pig liver ETF; reduction to the dihydroquinone level in pig liver ETF requires about 1 h for equilibration (13Gorelick R.J. Mizzer J.P. Thorpe C. Biochemistry. 1982; 21: 6936-6942Crossref PubMed Scopus (72) Google Scholar, 14Husain M. Steenkamp D.J. Biochem. J. 1983; 209: 541-545Crossref PubMed Scopus (65) Google Scholar). The potentials measured for free M. methylotrophus ETF (as with any ETF) may not of course reflect the situation in the electron transfer complex with its physiological electron donor (TMADH) but nevertheless are likely to serve as a reasonable guide. M. methylotrophus ETF shares considerable sequence identity with bacterial and mammalian ETFs (10Roberts D.L. Frerman F.E. Kim J.-J.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14355-14360Crossref PubMed Scopus (155) Google Scholar, 11Roberts D.L. Salazar D. Fulmer J.P. Frerman F.E. Kim J.J. Biochemistry. 1999; 38: 1977-1989Crossref PubMed Scopus (58) Google Scholar). Preliminary crystallographic studies of M. methylotrophus ETF have been reported (23White S.A. Mathews F.S. Rohlfs R.J. Hille R. J. Mol. Biol. 1994; 240: 265-266Crossref PubMed Scopus (10) Google Scholar), but to date no crystallographic structure for the protein is available. However, crystallographic structures of human andP. denitrificans ETF have been determined at 2.1 Å (10Roberts D.L. Frerman F.E. Kim J.-J.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14355-14360Crossref PubMed Scopus (155) Google Scholar) and 2.6 Å (11Roberts D.L. Salazar D. Fulmer J.P. Frerman F.E. Kim J.J. Biochemistry. 1999; 38: 1977-1989Crossref PubMed Scopus (58) Google Scholar) resolution, respectively. Using the x-ray structure of human ETF as a template (10Roberts D.L. Frerman F.E. Kim J.-J.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14355-14360Crossref PubMed Scopus (155) Google Scholar), we built a model of the structure ofM. methylotrophus ETF, in free solution and in complex with TMADH (19Chohan K.K. Scrutton N.S. Sutcliffe M.J. Protein Pept. Lett. 1998; 5: 231-236Google Scholar). The model predicts that the two subunits (subunit α (residues 1–321) and subunit β (residues 322–585)) of M. methylotrophus ETF comprise three domains. Domain I (the N-terminal region of the α subunit), domain II (the C terminus of the α subunit and a small C-terminal region of the β subunit), and domain III (the majority of the β subunit) form a Y-shaped structure, with domains I and III forming a shallow "bowl" in which domain II rests. Domain II is connected to domains I and III by two flexible regions of polypeptide chain (19Chohan K.K. Scrutton N.S. Sutcliffe M.J. Protein Pept. Lett. 1998; 5: 231-236Google Scholar). Small angle x-ray scattering studies have demonstrated that domain II is mobile with respect to domains I and III (20Jones M. Basran J. Sutcliffe M.J. Gunter Grossmann J. Scrutton N.S. J. Biol. Chem. 2000; 275: 21349-21354Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). The isoalloxazine ring of FAD interacts almost exclusively with domain II (Fig. 1). The model of M. methylotrophus ETF suggests that residue Arg-237 is located close to the FAD isoalloxazine ring, with its guanidinium group positioned over the si face of the dimethylbenzene subnucleus. The guanidinium group is thus located to help stabilize the increased electron density (which resides predominantly in the pyrimidine subnucleus) on reduction of the flavin to the anionic semiquinone. In this paper, we report the redox properties of a mutant ETF in which Arg-237 is replaced by Ala. We show that Arg-237 plays a key role in the exceptional stabilization of the anionic semiquinone in native ETF and that mutation of Arg-237 to Ala removes the kinetic block to full reduction of the FAD. These findings demonstrate that the chemical properties of a single residue close to the flavin isoalloxazine ring can have profound effects on the redox properties (thermodynamic and kinetic) of protein-bound flavin. Isolation of the αR237A mutant form of ETF was performed using the QuikChange site-directed mutagenesis kit supplied by Stratagene and oligonucleotides 5′-CTT TGC TGC TCA GCT CCG ATT GCG GAT-3′ and 5′-ATC CGC AAT CGG AGC TGA GCA GCA AAG-3′. The ETF expression plasmid pED1 (20Jones M. Basran J. Sutcliffe M.J. Gunter Grossmann J. Scrutton N.S. J. Biol. Chem. 2000; 275: 21349-21354Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) was used as template DNA for the mutagenesis reaction. Recombinant wild-type ETF was expressed from plasmid pED1 in E. coli strain TG1 as described (20Jones M. Basran J. Sutcliffe M.J. Gunter Grossmann J. Scrutton N.S. J. Biol. Chem. 2000; 275: 21349-21354Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). To ensure that no spurious changes had arisen as a result of the mutagenesis reaction, the entire ETF gene was resequenced using the Amersham Pharmacia Biotech T7 sequencing kit and protocols. Recombinant wild-type and mutant ETF proteins were expressed from plasmids pED1 and pED1R237A, respectively, in the E. colistrain TG1. Recombinant strains were grown at 20 °C in 2xYT medium supplemented with 100 μg/ml ampicillin. ETF was purified in large quantities (∼30 mg/liter of late exponential phase culture) from recombinant strains of E. coli. Harvested cells were resuspended in buffer A (50 mm potassium phosphate buffer, pH 7.2, 0.2 mm EDTA) and broken in a French press (140 megapascals, 4 °C). The extract was clarified by centrifugation at 15,000 × g for 90 min, and solid ammonium sulfate was added to 50% saturation. The precipitate was removed by centrifugation, and the supernatant was applied to a high performance phenyl-Sepharose column using a fast protein liquid chromatography system (Amersham Pharmacia Biotech) previously equilibrated with buffer A containing 1.5 m ammonium sulfate. After being washed with equilibration buffer, protein was eluted using a descending gradient (1.5 to 0 m) of ammonium sulfate. Fractions containing ETF were dialyzed exhaustively against buffer A and applied to a Q-Sepharose column equilibrated with buffer A. After washing with buffer A, protein was eluted using a gradient (0 to 2 m KCl); ETF was eluted at ∼0.5m KCl. Samples were dialyzed exhaustively against potassium phosphate buffer, pH 7.2 and stored (-70 °C) in the presence of 20% ethylene glycol. Redox titrations were performed within a Belle Technology glove box under a nitrogen atmosphere (oxygen maintained at <5 ppm) in 50 mm potassium phosphate buffer, pH 7.2. Anaerobic titration buffer was prepared by flushing freshly prepared buffer with oxygen-free nitrogen. Protein samples admitted to the glove box were deoxygenated by passing through a Bio-Rad 10DG column, with final dilution of the eluted protein to give an ETF concentration of 70–80 μm. Solutions of benzyl viologen, methyl viologen, 2-hydroxy-1,4-naphthaquinone, and phenazine methosulfate were added to a final concentration of 0.5 μm as redox mediators for the titrations. Absorption spectra (300–750 nm) were recorded on a Shimadzu 2101 UV-visible spectrophotometer, and the electrochemical potential was monitored using a CD 740 m combination pH/voltmeter coupled to a Russell platinum/calomel electrode. The electrode was calibrated using the Fe(II)/Fe(III)-EDTA couple (108 mV) as a standard. The flavoprotein solutions were titrated electrochemically using sodium dithionite as reductant and potassium ferricyanide as oxidant, as described by Dutton (24Dutton P. Methods Enzymol. 1978; 54: 422-435Google Scholar). After the addition of each aliquot of reductant, and allowing equilibration to occur (stabilization of the observed potential), the spectrum was recorded, and the potential was noted. The process was repeated at several (typically ∼40) different potentials. In this way, a set of spectra representing reductive and oxidative titrations was obtained. Small corrections were made for any drift in the base line by correcting the absorbance at 750 nm to zero, and spectrophotometric contributions from the mediators were removed prior to data analysis. The observed potentials were corrected to those for the standard hydrogen electrode (platinum/calomel + 244 mV). Data manipulation and analysis were performed using Origin software (Microcal). Absorbance values at wavelengths of 470 nm (near the oxidized flavin maximum) and 370 nm (close to the maximum for the anionic semiquinone) were plotted against potential. Data were fitted to Equation 1, which represents a two-electron redox process derived by extension to the Nernst equation and the Beer-Lambert law, as described previously (24Dutton P. Methods Enzymol. 1978; 54: 422-435Google Scholar, 25Daff S.N. Chapman S.K. Turner K.L. Holt R.A. Govindaraj S. Poulos T.L. Munro A.W. Biochemistry. 1997; 36: 13816-13823Crossref PubMed Scopus (207) Google Scholar). A=a10(E−E1′)/59+b+c10(E2′−E)/591+10(E−E1′)/59+10(E2′−E)/59Equation 1 In Equation 1, A is the total absorbance;a, b, and c are component absorbance values contributed by one flavin in the oxidized, semiquinone, and reduced states, respectively. E is the observed potential;E′1 and E′2 are the midpoint potentials for oxidized-semiquinone and semiquinone-reduced couples, respectively. In using Equation 1 to fit the absorbance-potential data, the variables were unconstrained, and regression analysis provided values in close agreement with those of the initial estimates. The αR237A mutant ETF was purified essentially as described for the wild-type protein. A notable difference between the mutant and wild-type proteins is the redox state of the flavin. αR237A ETF is purified in the oxidized form, whereas wild-type ETF is isolated as a mixture of oxidized and anionic flavin semiquinone forms (Fig. 2). Oxidation of the wild-type ETF with potassium ferricyanide, followed by immediate rapid gel filtration to remove the oxidant, generates the oxidized form of wild-type ETF. A comparison of the spectral properties of the oxidized wild-type and αR237A mutant ETF proteins indicates that the peak of flavin absorption (446 nm) in the αR237A mutant is shifted compared with the corresponding peak (438 nm) in wild-type ETF. In addition, the A388/A446ratio (1.01) for αR237A ETF is greater than theA380/A438 ratio (0.89) for wild-type ETF. These observations suggest that the isoalloxazine ring of FAD in αR237A ETF is more exposed to solvent than in wild-type ETF (26Muller F. Mayhew S.G. Massey V. Biochemistry. 1973; 12: 4654-4662Crossref PubMed Scopus (63) Google Scholar), an observation that is consistent with the structural model for M. methylotrophus ETF (19Chohan K.K. Scrutton N.S. Sutcliffe M.J. Protein Pept. Lett. 1998; 5: 231-236Google Scholar). Consistent with previous reports, anaerobic titration of wild-type ETF with sodium dithionite (16Davidson V.L. Husain M. Neher J.W. J. Bacteriol. 1986; 166: 812-817Crossref PubMed Google Scholar) or enzymatic reduction with TMADH (4Steenkamp D.J. Gallup M. J. Biol. Chem. 1978; 253: 4086-4089Abstract Full Text PDF PubMed Google Scholar) reduces the protein only to the level of the flavin anionic semiquinone (Fig. 3 A). The addition of excess dithionite (6× molar excess) does not reduce the protein further to the dihydroquinone form, even following prolonged incubation (30 min). The potential of the E′2 couple for wild-type ETF is almost certainly more negative than −250 mV (see below) but is more positive than the reduction potential of dithionite (-530 mV). This indicates that there is a substantial kinetic block on full reduction of the flavin. By contrast, reduction of the αR237A mutant ETF with dithionite proceeds to full reduction (Fig. 3, B and C). The two reductive phases (oxidized-semiquinone and semiquinone-dihydroquinone couples) are clearly resolved. The spectral changes accompanying reduction of the oxidized FAD to the anionic semiquinone have isosbestic points at 491 and 391 nm, and a single isosbestic point at 342 nm is seen for reduction of the anionic semiquinone to the dihydroquinone. In all cases, titrations were initiated from fully oxidized ETF and proceeded gradually to the end point of the titration by the addition of small aliquots of sodium dithionite (from 1 and 10 mm stocks) and then back again to oxidized ETF by addition of aliquots of potassium ferricyanide stocks of the same concentration. The protein samples remained completely soluble and stable throughout the course of the titration, enabling collection of good quality sets of spectra. No hysteretical effects were observed in any of the redox titrations. Spectra recorded at similar potentials during oxidative and reductive titrations were essentially identical. Representative spectra for the reductive titration of wild-type ETF and plots of the absorbanceversus potential are shown in Fig.4, A and B. In the reductive titration with wild-type ETF in the presence of mediators, reduction occurs first to the flavin anionic semiquinone, and a small proportion of the flavin is subsequently reduced to the dihydroquinone (Fig. 4 A). Partial reduction to the dihydroquinone does not occur in the absence of mediators (Fig. 3), with reduction taking place only to the level of the flavin anionic semiquinone; this requires further comment. The time taken to complete reductive titrations in the potentiometry experiments was typically around 3–4 h. This is clearly much longer than the time taken to complete the simple spectral analysis shown in Fig. 3. Redox mediators were also included in the potentiometric analysis but were absent in the spectroscopic characterization shown in Fig. 3. The presence of redox mediators and also the long time period for protein reduction ensured more complete equilibration of the system in the potentiometric analyses, but this is clearly not the case with the spectral changes displayed in Fig. 3. Notwithstanding the inclusion of redox mediators and the prolonged time periods of the potentiometry measurements, the spectral changes accompanying reduction of wild-type ETF in the presence of redox mediators indicates that completereduction to the dihydroflavin is not obtained. This is indicated by the presence of considerable semiquinone signature at 370 nm at the end of the titration. Further addition of dithionite did not reduce the absorption at 370 nm to the level seen for the αR237A mutant. That a substantial proportion of the wild-type ETF is reduced beyond the anionic semiquinone level, however, is indicated by the extent of bleaching in the absorption range of 440–470 nm (compared with titrations in the absence of mediators) and the partial bleaching of the absorption at 370 nm (Fig. 4, A and B). The inability to completely reduce wild-type ETF probably reflects the presence of a substantial kinetic block on full reduction for a proportion of the protein sample. Titrations performed in the presence and absence of mediators serve to illustrate the kinetic limitation on reduction to the dihydroquinone form in wild-type ETF, a kinetic bottleneck that can be overcome (at least in part) by the inclusion of redox mediators during the course of reductive titration. That equilibration was achieved with wild-type ETF in the majority of the potentiometric analyses is evident from the stability of the potential readings throughout the reductive titration and from the well defined transitions (Fig. 4 B). The lack of hysteresis on performing the oxidative titration likewise indicates that equilibration was achieved (except at very low potentials) and also that FAD was not released from ETF during the course of the potentiometric titrations. The midpoint reduction potentials forE′1 and E′2 were obtained by fitting the data shown in Fig. 4 A to Equation 1. These potentials are compared with values obtained by other workers for wild-type M. methylotrophus and mammalian ETFs in TableI.Table IMeasured midpoint reduction potentials for wild-type and αR237A M. methylotrophus (sp. W3A1) ETF and comparison with the midpoint reduction potentials of ETF proteins from other speciesSource of ETFMidpoint potential of E′1(oxidized-semiquinone) coupleMidpoint potential ofE′2 (semiquinone-dihydroquinone) couplemVmVM. methylotrophus wild type (this work) 1-aValues taken from the fit of Equation 1 to absorbance (470 nm) versus potential plots for wild-type and αR237A ETF proteins.+153 ± 2<−250M. methylotrophus αR237A (this work) 1-aValues taken from the fit of Equation 1 to absorbance (470 nm) versus potential plots for wild-type and αR237A ETF proteins.−43 ± 2−31 ± 2M. methylotrophus wild type (Ref.21Wilson E.K. Huang L. Sutcliffe M.J. Mathews F.S. Hille R. Scrutton N.S. Biochemistry. 1997; 36: 41-48Crossref PubMed Scopus (38) Google Scholar) 1-bValues determined using the xanthine/xanthine oxidase method of Massey (39).+141NDM. methylotrophus wild type (Ref. 17Byron C.M. Stankovich M.T. Husain M. Davidson V.L. Biochemistry. 1989; 28: 8582-8587Crossref PubMed Scopus (37) Google Scholar)+196−197Human wild type (Ref. 37Dwyer T. Zhang L. Muller M. Marrugo F. Frerman F. Biochim. Biophys. Acta. 1999; 1433: 139-152Crossref PubMed Scopus (18) Google Scholar) 1-bValues determined using the xanthine/xanthine oxidase method of Massey (39).+22−42Human αR249K (Ref.37Dwyer T. Zhang L. Muller M. Marrugo F. Frerman F. Biochim. Biophys. Acta. 1999; 1433: 139-152Crossref PubMed Scopus (18) Google Scholar) 1-bValues determined using the xanthine/xanthine oxidase method of Massey (39).−39−124Pig liver (Ref. 40Husain M. Stankovich M.T. Fox B.G. Biochem. J. 1984; 219: 1043-1047Crossref PubMed Scopus (34) Google Scholar)+4−50ND, not determined. A precise E′2 value for the wild-type M. methylotrophus ETF could not be obtained, because the flavin could not be converted completely to the dihydroquinone.1-a Values taken from the fit of Equation 1 to absorbance (470 nm) versus potential plots for wild-type and αR237A ETF proteins.1-b Values determined using the xanthine/xanthine oxidase method of Massey (39Massey V. Curti B. Ronchi S. Zanetti G. Flavins and Flavoproteins. Walter de Gruyter & Co., Berlin1991: 59-66Google Scholar). Open table in a new tab ND, not determined. A precise E′2 value for the wild-type M. methylotrophus ETF could not be obtained, because the flavin could not be converted completely to the dihydroquinone. As with wild-type ETF, αR237A ETF remained soluble throughout the course of reductive and oxidative titrations, and no hysteresis was observed during the course of reduction by dithionite and reoxidation by ferricyanide. Representative spectra during the course of reductive titration with dithionite are shown in Fig. 4 B, and plots of absorbance versus potential are shown in Fig.4 D. The spectral changes observed during reductive titration (over 3–4 h) are again different from those observed in the spectroscopic characterization of the αR237A ETF shown in Fig. 3, B andC. In Fig. 3, the anionic semiquinone species is populated prior to full reduction to the dihydroquinone, whereas in potentiometric titrations full development of the anionic semiquinone signature at 370 nm is not observed (Fig. 4,C and D). Again, we attribute this to a kinetic limitation that prevents rapid reduction to the dihydroquinone in the absence of mediators (see also the value for E′2below). However, mutation of αArg-237 to Ala partially relieves the kinetic block, because the dihydroquinone clearly does form in the αR237A mutant enzyme during the reductive titration performed without mediators (Fig. 3 C), unlike wild-type ETF, which is reduced only to the level of the flavin semiquinone (Fig. 3 A). We conclude, therefore, that αArg-237 contributes to the kinetic block on reduction to the dihydroquinone seen in wild-type ETF. During reductive titration of the αR237A mutant enzyme, small amounts of red anionic semiquinone are observed, as evidenced by very small increases in absorption at 370 nm during the early phase of reduction (Fig. 4 D). The data at this wavelength fit to a two-electron Nernst function, and the two one-electron reduction steps are resolved as semiquinone (E′1 (oxidized-semiquinone) = −43 ± 2 mV, with E′2(semiquinone-dihydroquinone) = −31 ± 2 mV). Clearl