Pallop Karnchanaphanurach

Electron transfer is used as a probe for angstrom-scale structural changes in single protein molecules. In a flavin reductase, the fluorescence of flavin is quenched by a nearby tyrosine residue by means of photo-induced electron transfer. By probing the fluorescence lifetime of the single flavin on a photon-by-photon basis, we were able to observe the variation of flavin-tyrosine distance over time. We could then determine the potential of mean force between the flavin and the tyrosine, and a correlation analysis revealed conformational fluctuation at multiple time scales spanning from hundreds of microseconds to seconds. This phenomenon suggests the existence of multiple interconverting conformers related to the fluctuating catalytic reactivity.

We report the results of a study, using digital video microscopy, of the melting transition in a quasi-two-dimensional suspension of uncharged silica spheres. This system was chosen to further test the dependence of the two-dimensional melting transition on the functional form of the colloid-colloid interaction. Our experimental data show that the solid phase undergoes a first order transition directly to the liquid phase. The system studied yields no evidence of the existence of a hexatic phase interpolating between the solid and liquid phases in the melting process.

Decay-accelerating factor (DAF, also known as CD55), a glycosylphosphatidylinositol-linked (GPI-linked) plasma membrane protein, protects autologous cells from complement-mediated damage by inhibiting complement component 3 (C3) activation. An important physical property of GPI-anchored complement regulatory proteins such as DAF is their ability to translate laterally in the plasma membrane. Here, we used single-particle tracking and tether-pulling experiments to measure DAF lateral diffusion, lateral confinement, and membrane skeletal associations in human erythrocyte membranes. In native membranes, most DAF molecules exhibited Brownian lateral diffusion. Fluid-phase complement activation caused deposition of C3b, one of the products of C3 cleavage, onto erythrocyte glycophorin A (GPA). We then determined that DAF, C3b, GPA, and band 3 molecules were laterally immobilized in the membranes of complement-treated cells, and GPA was physically associated with the membrane skeleton. Mass spectrometry analysis further showed that band 3, alpha-spectrin, beta-spectrin, and ankyrin were present in a complex with C3b and GPA in complement-treated cells. C3b deposition was also associated with a substantial increase in erythrocyte membrane stiffness and/or viscosity. We therefore suggest that complement activation stimulates the formation of a membrane skeleton-linked DAF-C3b-GPA-band 3 complex on the erythrocyte surface. This complex may promote the removal of senescent erythrocytes from the circulation.

Escherichia coli general NAD(P)H:flavin oxidoreductase (Fre) does not have a bound flavin cofactor; its flavin substrates (riboflavin, FMN, and FAD) are believed to bind to it mainly through the isoalloxazine ring. This interaction was real for riboflavin and FMN, but not for FAD, which bound to Fre much tighter than FMN or riboflavin. Computer simulations of Fre·FAD and Fre·FMN complexes showed that FAD adopted an unusual bent conformation, allowing its ribityl side chain and ADP moiety to form an additional 3.28 H-bonds on average with amino acid residues located in the loop connecting Fβ5 and Fα1 of the flavin-binding domain and at the proposed NAD(P)H-binding site. Experimental data supported the overlapping binding sites of FAD and NAD(P)H. AMP, a known competitive inhibitor with respect to NAD(P)H, decreased the affinity of Fre for FAD. FAD behaved as a mixed-type inhibitor with respect to NADPH. The overlapped binding offers a plausible explanation for the largeK m values of Fre for NADH and NADPH when FAD is the electron acceptor. Although Fre reduces FMN faster than it reduces FAD, it preferentially reduces FAD when both FMN and FAD are present. Our data suggest that FAD is a preferred substrate and an inhibitor, suppressing the activities of Fre at low NADH concentrations. Escherichia coli general NAD(P)H:flavin oxidoreductase (Fre) does not have a bound flavin cofactor; its flavin substrates (riboflavin, FMN, and FAD) are believed to bind to it mainly through the isoalloxazine ring. This interaction was real for riboflavin and FMN, but not for FAD, which bound to Fre much tighter than FMN or riboflavin. Computer simulations of Fre·FAD and Fre·FMN complexes showed that FAD adopted an unusual bent conformation, allowing its ribityl side chain and ADP moiety to form an additional 3.28 H-bonds on average with amino acid residues located in the loop connecting Fβ5 and Fα1 of the flavin-binding domain and at the proposed NAD(P)H-binding site. Experimental data supported the overlapping binding sites of FAD and NAD(P)H. AMP, a known competitive inhibitor with respect to NAD(P)H, decreased the affinity of Fre for FAD. FAD behaved as a mixed-type inhibitor with respect to NADPH. The overlapped binding offers a plausible explanation for the largeK m values of Fre for NADH and NADPH when FAD is the electron acceptor. Although Fre reduces FMN faster than it reduces FAD, it preferentially reduces FAD when both FMN and FAD are present. Our data suggest that FAD is a preferred substrate and an inhibitor, suppressing the activities of Fre at low NADH concentrations. NAD(P)H:flavin oxidoreductase riboflavin molecular dynamics potassium phosphate dithiothreitol electrospray ionization mass spectrometry high pressure liquid chromatography Escherichia coli general NAD(P)H:flavin oxidoreductase does not contain any bound flavin cofactor (1Fontecave M. Eliasson R. Reichard P. J. Biol. Chem. 1987; 262: 12325-12331Abstract Full Text PDF PubMed Google Scholar, 2Fieschi F. Nivière V. Frier C. Decout J.-L. Fontecave M. J. Biol. Chem. 1995; 270: 30392-30400Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). This property separates Fre1 from the flavin-containing NAD(P)H:flavin oxidoreductases of Vibrio harveyi and V. fischeri that supply FMNH2to bacterial luciferases (3Inouye S. FEBS Lett. 1994; 347: 163-168Crossref PubMed Scopus (64) Google Scholar, 4Lei B. Liu M. Huang S. Tu S.C. J. Bacteriol. 1994; 176: 3552-3558Crossref PubMed Google Scholar). Fre uses either NADH or NADPH as electron donors to reduce FAD, FMN, or riboflavin (Rfl); however, when FAD is the electron acceptor, the K m values for NADH and NADPH are exceptionally large (1Fontecave M. Eliasson R. Reichard P. J. Biol. Chem. 1987; 262: 12325-12331Abstract Full Text PDF PubMed Google Scholar, 2Fieschi F. Nivière V. Frier C. Decout J.-L. Fontecave M. J. Biol. Chem. 1995; 270: 30392-30400Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 5Nivière V. Fieschi F. Decout J.-L. Fontecave M. J. Biol. Chem. 1999; 274: 18252-18260Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Reduced flavins generated by Fre are believed to have important biological functions. It has been shown that the reduced flavins generated by Fre can regulate the activity of the aerobic ribonucleotide reductases by regenerating or scavenging the Tyr122 radical of the ribonucleotide reductase in vitro (1Fontecave M. Eliasson R. Reichard P. J. Biol. Chem. 1987; 262: 12325-12331Abstract Full Text PDF PubMed Google Scholar, 6Fontecave M. Eliasson R. Reichard P. J. Biol. Chem. 1989; 264: 9164-9170Abstract Full Text PDF PubMed Google Scholar). An E. coli fremutant is more susceptible to hydroxyurea, a scavenger of the Tyr122 radical, than the wild-type E. coli (7Covès J. Nivière V. Eschenbrenner M. Fontecave M. J. Biol. Chem. 1993; 268: 18604-18609Abstract Full Text PDF PubMed Google Scholar), pointing to the protective role of Fre for the aerobic ribonucleotide reductase in vivo. Fre produces reduced flavins that can reduce metal ions, including ferrisiderophores (8Covès J. Fontecave M. Eur. J. Biochem. 1993; 211: 635-641Crossref PubMed Scopus (95) Google Scholar), Cob(III)alamin (9Fonseca M. Escalante-Semerena J.C. J. Bacteriol. 2000; 182: 4304-4309Crossref PubMed Scopus (57) Google Scholar), and chromate (10Puzon G.J. Petersen J.N. Roberts A.G. Kramer D.M. Xun L. Biochem. Biophys. Res. Commun. 2002; 294: 76-81Crossref PubMed Scopus (118) Google Scholar). Fre is capable of supplying FADH2 to the FADH2-utilizing monooxygenases (11Xun L. Sandvik E.R. Appl. Environ. Microbiol. 2000; 66: 481-486Crossref PubMed Scopus (90) Google Scholar, 12Louie T.M. Webster C.M. Xun L. J. Bacteriol. 2002; 184: 3492-3500Crossref PubMed Scopus (111) Google Scholar). Despite these apparent biological functions, the genuine physiological role of Fre remains unclear. Recently, Ingelman et al. (13Ingelman M. Ramaswamy S. Nivière V. Fontecave M. Eklund H. Biochemistry. 1999; 38: 7040-7049Crossref PubMed Scopus (77) Google Scholar) reported the crystal structure of Fre with or without bound Rfl, revealing that Fre is similar to members of the ferredoxin:NADP+ reductase family in structure, although the similarities in amino acid sequence between Fre and members of the ferredoxin:NADP+ reductase family are low. The crystal structure shows that Fre is organized into an N-terminal flavin-binding domain and a C-terminal NAD(P)H-binding domain; the secondary structures of the two domains are labeled as F and N, respectively. The most interesting feature is that the loop connecting Fβ5 and Fα1 in the flavin-binding domain that normally interacts with the ADP moiety of FAD in other ferredoxin:NADP+ reductases is exceptionally short in Fre (13Ingelman M. Ramaswamy S. Nivière V. Fontecave M. Eklund H. Biochemistry. 1999; 38: 7040-7049Crossref PubMed Scopus (77) Google Scholar). Consequently, although FAD is a bound cofactor in most ferredoxin:NADP+ reductase proteins, all three flavin substrates of Fre do not remain bound (13Ingelman M. Ramaswamy S. Nivière V. Fontecave M. Eklund H. Biochemistry. 1999; 38: 7040-7049Crossref PubMed Scopus (77) Google Scholar). Because the crystal structure of the Fre·Rfl complex revealed the interactions between the isoalloxazine ring of Rfl and the flavin-binding domain (12Louie T.M. Webster C.M. Xun L. J. Bacteriol. 2002; 184: 3492-3500Crossref PubMed Scopus (111) Google Scholar) and because the K m values of Fre for Rfl, FMN, and FAD are very similar (2Fieschi F. Nivière V. Frier C. Decout J.-L. Fontecave M. J. Biol. Chem. 1995; 270: 30392-30400Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), it has been proposed that all three flavins mainly interact with residues of the flavin-binding domain through the isoalloxazine ring. However, direct experimental data are unavailable to support the proposed interactions of Fre with FMN and FAD. In this report we characterized FAD and FMN binding to Fre. TheK d values of Fre with FAD and FMN were determined. The value with FAD was much smaller than that with FMN or Rfl. Molecular dynamics (MD) simulations of Fre with FAD predicted that the ribityl side chain and the ADP moiety of FAD confer extra stability to the Fre·FAD complex. Experimental data supported the simulation model. Studies on the physiological roles of the high affinity of Fre for FAD revealed that FAD is a preferred substrate and inhibitor of Fre. An in vivo model for regulating Fre activities in responding to O2 supply is proposed. All reagents were of the highest purity available and were purchased from Sigma, Aldrich, or Fisher Scientific Co. HpaB, an FADH2-utilizing 4-hydroxyphenylacetate 3-monooxygenase, was overproduced and purified from E. coliBL21(DE3) carrying pES2 (11Xun L. Sandvik E.R. Appl. Environ. Microbiol. 2000; 66: 481-486Crossref PubMed Scopus (90) Google Scholar). Overexpression of the clonedfre gene in E. coli BL21(DE3)(pES1) and purification of Fre were done primarily as reported previously (11Xun L. Sandvik E.R. Appl. Environ. Microbiol. 2000; 66: 481-486Crossref PubMed Scopus (90) Google Scholar). To ensure Fre of the highest purity, a phenyl-agarose chromatography was added to the previously reported procedures. The ammonium sulfate precipitated proteins were dissolved in 20 mm KPi buffer (pH 7.0) containing 1 mm DTT with 25% saturation of ammonium sulfate and loaded onto a phenyl-agarose (Sigma) column (1.5 × 12.5 cm) equilibrated with the same buffer. The proteins were eluted with a linear gradient of ammonium sulfate (25% to 0%, 200 ml) in KPi buffer with 1 mm DTT at a flow rate of 1 ml·min−1. Active fractions eluted with ∼10% saturation of ammonium sulfate were pooled together and dialyzed against several changes of 20 mm KPi buffer (pH 7.0) with 1 mm DTT overnight. The sample was then purified by going through a Bioscale Q column and Superdex 75 column as reported previously (11Xun L. Sandvik E.R. Appl. Environ. Microbiol. 2000; 66: 481-486Crossref PubMed Scopus (90) Google Scholar). Purified protein was analyzed by SDS-PAGE (14Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207018) Google Scholar). ESI-MS was done with a Waters Micromass ZQ mass spectrometer. Fre samples in 10 mm Tris-HCl buffer (pH 7.5) were acidified with formic acid to a final concentration of 0.1% (v/v) and infused into the electrospray ionization source at a flow rate of 10 μl·min−1. The electrospray ionization source temperature and the desolvation temperature were maintained at 90 °C and 150 °C, respectively. The capillary voltage and the cone voltage were 3400 V and 35 V, respectively. Spectra were scanned fromm/z 800 to 3000 at a rate of 1 scan/s. Protein mass spectrum was deconvoluted using the MaxEnt software (Waters). NAD(P)H:flavin oxidoreductase activity was determined by the of NADH in 20 buffer (pH 7.0) containing NADH and 10 FMN or FAD at of NAD(P)H:flavin oxidoreductase activity was as the of 1 was with and 10 in 20 of 20 mm KPi buffer (pH 7.0) containing NADH and with either of FAD and FMN or FAD. The was at °C for and the of was by a previously reported (11Xun L. Sandvik E.R. Appl. Environ. Microbiol. 2000; 66: 481-486Crossref PubMed Scopus (90) Google Scholar). with a and a column × was to the of Fre that bound Fre was eluted from the column with an flow of 20 mm KPi (pH 7.0) buffer with 1 mm DTT at ml·min−1. The of flavin with Fre was by the of Fre at with the of flavin The same with a column × 150 was to the of the bound Fre sample was eluted from the column with a linear gradient of in 0.1% acid with a flow rate of 1 ml·min−1. of the flavin from Fre was with of flavin The K of the Fre·FAD and Fre·FMN complexes were determined by a using a Fre in 20 buffer (pH was with of flavin added from a and the in of flavin was The was at and of Fre was from to at and were at The of Fre was against the of concentration of FAD to Fre and with the 1 in which is a to the model with experimental is the of is the concentration of Fre at that 1 is the of the Fre·FAD and is the final concentration of the Fre·FAD complex. The value is by the The of on the K d of Fre for FAD was determined. The experimental was to that that mm was in the The of the Fre·FAD and Fre·FMN complexes were on the crystal structure of the Fre·Rfl complex Nivière of the The protein sequence was and by simulations using the R. Commun. 1995; Scopus Google Scholar, E. B. J. Google Scholar) on a simulations were in the of of as were for the simulation The was for the Fre·FMN the was for the Fre·FAD the and the was to for 800 with temperature and pressure to an was for of the Fre·FAD and Fre·FMN between Fre and the flavin substrate were is when the between the and and and is than was the to the structure for the In the a structure is added to the when its to any of the is than the The structures were to structure from the of the was from the as the The of FAD on Fre activity were in the of NADPH and were against the substrate or at of FAD to the of the were then determined from the with competitive and using the The apparent of Fre for the substrate were determined from with the using the of Fre was purified from of protein in the The protein was purified to apparent The purified Fre was acidified and analyzed by the molecular of Fre was determined to which is to molecular of from the amino acid other molecular mass was by the purity of the Fre The purified Fre a activity of when FMN with NADH as the electron at The and Fre a the of flavins bound to Fre. The flavin bound to Fre was 200 of Fre was loaded onto an we a of flavin with the Fre on the spectrum by the The flavin was at the of the protein with the molecular of the complex as with that of Fre the of the flavin that with Fre and it with the of FMN data showed that of FMN was with Fre. Fre was loaded onto a the flavin was from the with a of spectrum of a flavin was The of FAD, FMN, and Rfl were and that the flavin is FAD. This was by of a of Fre with either FMN or FAD onto the 25% of FAD in the was with but of FMN in the was with of Fre by a Fre sample was eluted from the column with a linear gradient of in 0.1% acid with a flow rate of 1 ml·min−1. with at was eluted from the column at a with at was eluted from the column at spectrum of the with at and of FAD were added to a Fre in 20 mm KPi (pH buffer. The binding of FAD to Fre the of in a in the of Fre The K d for FAD was determined to the of Fre against the of the added FAD concentration to the Fre concentration a similar the K d for FMN was determined to The d value for FAD is not with the that the isoalloxazine of the flavin substrates the for the binding of the three flavins to Fre and that the flavins have similar affinity for Fre (13Ingelman M. Ramaswamy S. Nivière V. Fontecave M. Eklund H. Biochemistry. 1999; 38: 7040-7049Crossref PubMed Scopus (77) Google Scholar). that the ribityl moiety and the ADP moiety of FAD interact with other in Fre and extra stability for the Fre·FAD complex. To this the structure of the Fre·FAD complex was by simulations showed that FAD an unusual bent acid residues located in the loop connecting Fβ5 and Fα1 in the flavin-binding domain and and at the proposed NAD(P)H-binding and were shown to form additional with the ribityl side chain and the ADP moiety of FAD the showed that were of a with between the Fre protein and the FAD were between Fre and The of to with the of the Fre·FAD complex. This is in in which we two of the FAD. The of the Fre·FAD complex that the ADP moiety of FAD interacts with amino acid residues located at the proposed NAD(P)H-binding site. This was TheK d value for FAD was determined to in the of mm AMP, a known competitive inhibitor of Fre with respect to values from to (2Fieschi F. Nivière V. Frier C. Decout J.-L. Fontecave M. J. Biol. Chem. 1995; 270: 30392-30400Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 5Nivière V. Fieschi F. Decout J.-L. Fontecave M. J. Biol. Chem. 1999; 274: 18252-18260Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). This K d value is than the K d value determined in the of The of FAD as an inhibitor for Fre activity was using NADPH as the electron and FMN as the electron acceptor. It has been reported that the K of Fre is with a of FAD is the electron V. Fieschi F. Decout J.-L. Fontecave M. J. Biol. Chem. 1999; 274: 18252-18260Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Because the highest concentration of NADPH in was Fre not have any activity for FAD to the large K Because FAD was not a substrate for Fre the its on FMN with NADPH was Fre activity was determined as a of NADPH concentration in the of a concentration of FMN at several of FAD. The showed a of at a to the of the that FAD is a mixed-type inhibitor with respect to NADPH with a K and a of the apparent K was Fre activity was determined as a of FMN concentration in the of a concentration of NADPH at several of FAD, FAD a competitive with a K of and an apparent K of The of FAD on the activity of Fre for FMN with NADH as the electron was Because FAD by Fre is much than FMN when NADH is than V. Fieschi F. Decout J.-L. Fontecave M. J. Biol. Chem. 1999; 274: 18252-18260Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), the of FAD was by the Fre activity a concentration of NADH and FMN with FAD concentrations. FAD at 1 reduced the rate of NADH of Fre by and at 10 FAD reduced the rate by The was shown to to the of FAD when the of FADH2 was by HpaB, an FADH2-utilizing 4-hydroxyphenylacetate that does not (11Xun L. Sandvik E.R. Appl. Environ. Microbiol. 2000; 66: 481-486Crossref PubMed Scopus (90) Google Scholar). was in to ensure of FADH2 in the a of NADH was and of three samples of was with FAD in the of the was in the of of FMN and FAD. of the NADH was to reduce FAD the containing of FAD and FMN, with the for FMN Fre a much affinity for FAD than for TheK d value for the Fre·FAD complex was than that of FMN (1.5 The K d values for Rfl and a flavin are and V. Fieschi F. Decout J.-L. Fontecave M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, V. Fontecave M. Biochemistry. PubMed Scopus Google Scholar). its flavin Fre FAD the most The binding was by the of FAD with Fre from the chromatography of the simulations showed that not the isoalloxazine ring of FAD but the ribityl side chain and the ADP moiety were in binding to support for tighter binding for FAD than for FMN of Rfl. the of the Fre·Rfl structure, which shows direct from the of the ribityl side chain to the of (13Ingelman M. Ramaswamy S. Nivière V. Fontecave M. Eklund H. Biochemistry. 1999; 38: 7040-7049Crossref PubMed Scopus (77) Google Scholar), and the similar K m values for Rfl, FMN, and FAD, it has been that Fre Rfl, FMN, and FAD mainly through the isoalloxazine ring (1Fontecave M. Eliasson R. Reichard P. J. Biol. Chem. 1987; 262: 12325-12331Abstract Full Text PDF PubMed Google Scholar, M. Ramaswamy S. Nivière V. Fontecave M. Eklund H. Biochemistry. 1999; 38: 7040-7049Crossref PubMed Scopus (77) Google Scholar). The proposed interaction is real for Rfl and FMN, but not for FAD. The simulations predicted the of 3.28 extra H-bonds on average from the ribityl side chain and ADP moiety of FAD with Fre the of a to Biochemistry. Scholar), the extra H-bonds in the Fre·FAD complex to a of in with the Fre·FMN complex. This as an because the bent and of the FAD can the of the Fre·FAD reduce the This is with the extra of from the determined K d values of the Fre·FAD complex to the Fre·FMN complex. The bent of FAD predicted by simulations is similar to the adopted by the FAD in the E. coli in which FAD a M. V. Eklund H. J. Biol. 268: PubMed Scopus Google Scholar). the reductase a loop connecting Fβ5 and as does Fre. sequence of Fre with the reductase shows that two reductase residues that with the ribityl side chain and the ring of the FAD and are in Fre and (13Ingelman M. Ramaswamy S. Nivière V. Fontecave M. Eklund H. Biochemistry. 1999; 38: 7040-7049Crossref PubMed Scopus (77) Google Scholar, M. V. Eklund H. J. Biol. 268: PubMed Scopus Google Scholar). The of with FAD is by of simulations the interaction of with FAD is not in the The reductase has a at the which additional interaction with the ring of FAD. a is not in the Fre and Fre does not bind FAD the reductase the flavin-binding the NAD(P)H-binding of Fre is a proposed model (13Ingelman M. Ramaswamy S. Nivière V. Fontecave M. Eklund H. Biochemistry. 1999; 38: 7040-7049Crossref PubMed Scopus (77) Google Scholar) on with ferredoxin:NADP+ complexes L. M. C. J.C. M. J. Biol. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). The ring and the of the moiety of NADPH are proposed to bind to Fre the amino of the of the NAD(P)H-binding and and bind the of (13Ingelman M. Ramaswamy S. Nivière V. Fontecave M. Eklund H. Biochemistry. 1999; 38: 7040-7049Crossref PubMed Scopus (77) Google Scholar). simulations predicted amino acid residues at these as and to form H-bonds with the ADP moiety of FAD The overlapping binding sites of FAD and were supported by several of a known competitive inhibitor with respect to the (2Fieschi F. Nivière V. Frier C. Decout J.-L. Fontecave M. J. Biol. Chem. 1995; 270: 30392-30400Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 5Nivière V. Fieschi F. Decout J.-L. Fontecave M. J. Biol. Chem. 1999; 274: 18252-18260Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), the K d value of the Fre·FAD complex from to In or an NADPH has on the K d of Fre for Rfl V. Fontecave M. Biochemistry. PubMed Scopus Google Scholar). the binding of FAD to Fre by with the ADP moiety of FAD for the NAD(P)H-binding to an K showed that FAD as a competitive inhibitor with respect to FMN and a mixed-type inhibitor with respect to NADPH of FAD FMN is because FAD with FMN for the flavin-binding site. However, the mixed-type NADPH the simulation model. an inhibitor with respect to Rfl, is an inhibitor with respect to NADPH (2Fieschi F. Nivière V. Frier C. Decout J.-L. Fontecave M. J. Biol. Chem. 1995; 270: 30392-30400Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). does not the of complex. The interactions between the ADP moiety of FAD and residues at the NAD(P)H-binding the of to the mixed-type The bent of FAD a plausible explanation for the large K and K values FAD in with the m values for Rfl and FMN (1Fontecave M. Eliasson R. Reichard P. J. Biol. Chem. 1987; 262: 12325-12331Abstract Full Text PDF PubMed Google Scholar, 2Fieschi F. Nivière V. Frier C. Decout J.-L. Fontecave M. J. Biol. Chem. 1995; 270: 30392-30400Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 5Nivière V. Fieschi F. Decout J.-L. Fontecave M. J. Biol. Chem. 1999; 274: 18252-18260Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Because FAD to Fre through both the isoalloxazine ring and the ADP moiety NADH has to with the ADP moiety of FAD for the binding site. Consequently, a concentration of NADH is to the ADP moiety of FAD for the NAD(P)H-binding to a K value than that for Rfl and FMN In the bent conformation, the of FAD is located in to the proposed binding for the of NADPH (13Ingelman M. Ramaswamy S. Nivière V. Fontecave M. Eklund H. Biochemistry. 1999; 38: 7040-7049Crossref PubMed Scopus (77) Google Scholar) The the binding of NADPH through with the of NADPH. value is than when FAD is the electron acceptor. The phosphate of FMN NADPH binding to Fre by the same in a K of from is FMN not The high affinity of Fre for FAD that the in activity is much than previously aerobic NADH and NADPH coli have been reported in the of 20 and 150 Escherichia coli and and Molecular Scholar). Rfl, FMN, and FAD have not been the FAD concentration of to M. H. H. M. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). Rfl concentration much than of FMN and FAD because Rfl into the FMN and FAD in to its S. Escherichia coli and and Molecular Scholar). the FMN and FAD to in the of then in vivo Fre activity mainly to the of FAD by NADH as by in vitro and the Fre NADPH to reduce FAD in vivo because value is much than the NADPH the in vivo Fre activity to the low NADH concentration in with the K The low in activity is to the because this of of the and of as Biochem. J. PubMed Scopus Google Scholar). In this that FAD more to Fre than either FMN or Rfl The binding FAD the preferred substrate in vivo Experimental data and simulations that the binding is to an unusual bent of FAD, allowing additional interactions between FAD and Fre. The ADP moiety of FAD with for binding to the largeK m values for NADH and NADPH FAD coli is a both and E. coli with O2 the NADH concentration is 20 or of NADH and Escherichia coli and and Molecular Scholar). The low NADH concentration very low Fre activity for FAD and FAD FMN by Fre. data suggest that Fre activities are in E. coli Nivière of the for Liu of for in the ESI-MS and M. Webster for in