Formation of W(3)A(1) electron-transferring flavoprotein (ETF) hydroquinone in the trimethylamine dehydrogenase x ETF protein complex.

The electron-transferring flavoprotein (ETF) from Methylophilus methylotrophus (sp. W(3)A(1)) exhibits unusual oxidation-reduction properties and can only be reduced to the level of the semiquinone under most circumstances (including turnover with its physiological reductant, trimethylamine dehydrogenase (TMADH), or reaction with strong reducing reagents such as sodium dithionite). In the present study, we demonstrate that ETF can be reduced fully to its hydroquinone form both enzymatically and chemically when it is in complex with TMADH. Quantitative titration of the TMADH x ETF protein complex with sodium dithionite shows that a total of five electrons are taken up by the system, indicating that full reduction of ETF occurs within the complex. The results indicate that the oxidation-reduction properties of ETF are perturbed upon binding to TMADH, a conclusion further supported by the observation of a spectral change upon formation of the TMADH x ETF complex that is due to a change in the environment of the FAD of ETF. The results are discussed in the context of ETF undergoing a conformational change during formation of the TMADH x ETF electron transfer complex, which modulates the spectral and oxidation-reduction properties of ETF such that full reduction of the protein can take place.

molecules per dimer. FAD is the only oxidation-reduction center in these proteins, although the ETFs from human, pig, P. denitrificans, and M. methylotrophus also contain one equivalent of AMP of unknown function (9 -12). Unlike most flavoproteins, which function as two-electron carriers, most ETFs are thought to be one-electron carriers, cycling between the oxidized and anionic flavin semiquinone (1). Nevertheless, it has been shown that mammalian ETFs and ETF from P. denitrificans can become fully reduced to the hydroquinone upon dithionite or photochemical reductions, although the reduction from semiquinone to hydroquinone is quite sluggish (4,6,13,14).
ETF from M. methylotrophus W 3 A 1 is the physiological electron acceptor of trimethylamine dehydrogenase (TMADH), an iron-sulfur-containing flavoprotein that catalyzes the oxidative demethylation of trimethylamine (8,15,16). The protein is a 63-kDa heterodimer with a 33.7-kDa ␣-subunit and a 28.9-kDa ␤-subunit (17); it contains one equivalent each of FAD and AMP per dimer. Unlike mammalian ETFs, which can accept reducing equivalents from a variety of electron donors, including at least nine different dehydrogenases in the mitochondria (18), W 3 A 1 ETF is highly specific for TMADH and cannot be reduced by other dehydrogenases from M. methylotrophus, such as methylamine dehydrogenase and methanol dehydrogenase (8), or by dehydrogenases from other organisms (19). During turnover, W 3 A 1 ETF receives a single reducing equivalent from the 4Fe/4S center of TMADH: the anionic semiquinone form of the protein rapidly accumulates in both steadystate (8,19) and rapid reaction experiments (20).
ETFs are distinct from most other flavoproteins in that the (anionic) semiquinone form is extremely stable (1). In the case of W 3 A 1 ETF, even strong reducing reagents such as dithionite and deazariboflavin radical are unable to reduce the protein to its two-electron reduced hydroquinone form (8,19). Full reduction of W 3 A 1 ETF has only been achieved by electrochemical means using reduced methylviologen as a redox mediator, with an equilibration time of 6 -12 h required for the transfer of the second electron (21). The mid-point reduction potentials obtained for the FAD quinone/semiquinone and semiquinone/ hydroquinone couples in this study were 196 mV and Ϫ197 mV, respectively. The unexceptional reduction potential for the semiquinone/hydroquinone couple (Ϫ197 mV) indicates that the barrier to full reduction of W 3 A 1 ETF is kinetic rather than thermodynamic in nature, as reduction cannot be achieved by either sodium dithionite (Ϫ530 mV) or photochemically generated deazariboflavin radical (Ϫ650 mV). These reduction potentials indicate, however, that electron transfer from neither the FMN cofactor (E 0Ј FMN/FMNsq ϭ 44 mV; E 0Ј FMNsq/FMNhq ϭ 36 mV) nor the 4Fe/4S center (E 0Ј ϭ 100 mV) (22) of TMADH to the ETF semiquinone is thermodynamically favorable.
The above discussion notwithstanding, in examining the re-action of the TMADH⅐ETF protein complex with trimethylamine, we have found unexpectedly that W 3 A 1 ETF can be fully reduced to the hydroquinone within the complex. Dithionite titration of the complex indicates that a total of five electrons are taken up, consistent with full reduction of ETF along with TMADH in the complex. Full reduction of W 3 A 1 ETF takes place via the 4Fe/4S center, as reduced phenylhydrazine-inactivated TMADH (in which the enzyme flavin has been covalently modified and rendered redox-inert; Ref. 23) remains capable of reducing ETF from the semiquinone to the hydroquinone state to a substantial degree. The results suggest that the oxidation-reduction properties of ETF are perturbed upon binding to TMADH, and specifically that the apparent kinetic barrier for reduction to the ETF hydroquinone has been overcome in the protein-protein complex. Electron transfer from TMADH to ETF has been studied previously by following the reaction of reduced TMADH with oxidized ETF, yielding a limiting rate constant of 172 s Ϫ1 and a dissociation constant for TMADH red and ETF ox of 10 mM (20). The reaction of substrate-reduced TMADH with the artificial electron acceptor ferricenium hexafluorophosphate has also been studied (24). The limiting rate constant for electron transfer as a function of temperature was analyzed by electron transfer theory, and an electron tunneling pathway distance of approximately 13 Å was obtained, which correlates with the shortest pathway measured from the 4Fe/4S center to the protein surface of TMADH. This effective electron transfer distance was further refined to 11.3 Å by analyzing the reaction of dithionite reduced phenylhydrazine-inactivated TMADH with ETF; based on the x-ray crystal structure of TMADH and the kinetically determined electron tunneling distance, it was proposed that electrons are transferred to the surface of TMADH at or close to residue Tyr-442 (25). The implication is that ETF docks close to Tyr-442 in forming the protein-protein complex. Kinetic studies of TMADH mutant proteins (Y442F, Y442L, and Y442G) have shown that Tyr-442 indeed plays a significant role in facilitating electron transfer from TMADH to ETF (25).
Given the observations described above, a structural model for the TMADH⅐ETF complex has been proposed (26). On the basis of this computer-modeled structure, a substantial conformational change of W 3 A 1 ETF during complex formation must take place for efficient electron transfer from the 4Fe/4S center of TMADH to the FAD cofactor of ETF (26). It has also been suggested that such a conformational change alters the environment of the FAD binding pocket and modulates the reduction potential of the cofactor. In light of the present work, it appears likely that the proposed conformational change indeed occurs, altering the oxidation-reduction properties of ETF such that its full reduction can take place.
Protein Purification and Sample Preparation-M. methylotrophus W 3 A 1 was grown on TMA as sole carbon source, and both TMADH and ETF were purified as described previously (20,28). Concentrated protein stock solutions were stored in liquid nitrogen with 15% glycerol added. All reactions were performed in 0.1 M potassium phosphate buffer, pH 7.0, at room temperature unless specified otherwise. Protein samples for the experiments were prepared in the following way. For TMADH, the thawed, concentrated enzyme stock solution was passed through a Sephadex G-25 column to remove glycerol and the concentration determined from the absorbance at 442 nm of oxidized enzyme (using an extinction coefficient of 27.3 mM Ϫ1 cm Ϫ1 ; Ref. 29). In the case of ETF, which is partially reduced as isolated, the protein was reoxi-dized with excess ferricenium hexafluorophosphate and then passed through a Sephadex G-25 column to remove residual oxidant. The concentration of ETF was determined from the 438-nm absorbance of oxidized protein using an extinction coefficient of 11.3 mM Ϫ1 cm Ϫ1 (8). When necessary, protein solutions were concentrated using Amicon Centricon-30 and Centricon-10 concentrators for TMADH and ETF, respectively.
The complex of TMADH with ETF was prepared simply by mixing high concentrations of the two proteins in 1:1 stoichiometry, given the small value expected for the K d between the oxidized forms of the two proteins (a K d of 10 M has been determined kinetically for complex formation between reduced TMADH and oxidized ETF; Ref. 20). Theoretically, up to millimolar concentrations of the two proteins can be used to ensure nearly complete formation of the protein complex. However, the maximum concentrations used in the experiments were limited by the 1-cm light path of the optical cell and the non-linearity of Beer's law at high optical density. Therefore, 100 M amounts of each protein were used in most cases, which affords 73% complex formation (based on a K d of 10 M). At 35 M for both proteins, on the other hand, it is estimated that about 59% of the proteins will be present as complex.
Treatment of TMADH with Phenylhydrazine Hydrochloride-Phenylhydrazine-inactivated TMADH was prepared based on the methods described previously (23,29). 50 M TMADH in 0.1 M sodium pyrophosphate buffer, pH 7.7, was incubated with 2 mM phenylhydrazine at 30°C for 4 h. The solution was then concentrated using Amicon Centricon-30 and passed through a Sephadex G-25 column to remove excess phenylhydrazine. The concentration of TMADH thus obtained was determined using an extinction coefficient of 21.4 mM Ϫ1 cm Ϫ1 at 410 nm (29).
UV-visible Spectroscopy-UV-visible spectra were recorded at room temperature using a Hewlett-Packard 8452A single-beam diode array spectrophotometer. In the reaction of TMADH⅐ETF with TMA, 300 l of the TMADH⅐ETF complex solution (100 M each) was placed in a 1-mm light path cell and the spectrum recorded. 10 l of a 18.6 mM TMA solution was then added to the sample to obtain a final concentration of 600 M. The cell was quickly mixed after the addition of TMA and spectra recorded every minute for 10 min. Spectral change associated with the initial reduction of the protein complex as well as spectral change occurring subsequent to the addition of TMA were obtained by simple spectral subtraction methods using Hewlett-Packard software.
For the reaction of three-electron reduced TMADH (TMADH 3eϪ ) with ETF semiquinone (ETF sq ), protein solutions were prepared in the following way; a 70 M TMADH solution containing 0.5 mM benzyl viologen (to prevent formation of bisulfite adduct at the FMN site of TMADH during dithionite titration; Ref. 30) was placed in a tonometer, made anaerobic by repeated evacuation and flushing with O 2 -free argon, then titrated to three-electron reduced level with a concentrated anaerobic sodium dithionite solution prepared in the same buffer. A solution of ETF (70 M) was placed in another tonometer, made anaerobic using the procedures described above, and titrated with sodium dithionite solution to the one-electron reduced level. 1.2 ml of each protein solution was then transferred into separate compartments of a split-cell cuvette (0.5-cm light path for each compartment) fitted with a rubber septum (which had been previously made anaerobic by flushing with O 2 -free argon) using a long-needle Hamilton syringe. The spectrum of the sample with TMADH 3eϪ and ETF sq in separate compartments was recorded; the split cell was then tipped, and the two solutions were mixed well and another spectrum recorded. Spectra of the samples were then measured every 20 s for 15 min.
For the reaction of reduced phenylhydrazine-inactivated TMADH with ETF sq , the two protein solutions were prepared in a similar way and the experiment performed essentially as described above. In this case, one compartment of the split cell contained reduced phenylhydrazine-inactivated TMADH rather than fully reduced TMADH.
The spectral change associated with binding of oxidized ETF and TMADH was obtained in the following way; 1.2 ml of 80 M oxidized ETF and TMADH were placed into separate compartments of a split cell cuvette. A spectrum of the sample was recorded (before mixing). The two solutions were then mixed thoroughly and another spectrum recorded (after mixing). The difference spectrum ((after mixing) Ϫ (before mixing)) was then obtained by spectral subtraction.
Sodium Dithionite Titration-A 1-ml solution of TMADH⅐ETF (100 M each) was placed in a tonometer equipped with a 4-mm light path cuvette and made anaerobic by repeated evacuation and flushing with O 2 -free argon. The protein solution was then titrated with a concentrated solution of sodium dithionite prepared in anaerobic buffer. The spectrum of the sample was recorded after each addition of sodium dithionite (spectra were continuously taken after adding the reductant until the system reached a new equilibrium; the spectrum of the sample at equilibrium required less than 1 min at the beginning and about 5 min toward the end of titration, and was used for the final plot), and the titration was considered complete when no further spectral change was observed with further addition of the reducing agent. After the titration of the protein complex was complete, the same dithionite solution was then calibrated immediately by titration against a 1-ml solution of anaerobic FMN of known concentration to determine the reductive strength of the sodium dithionite solution. The A 370 from the titration with the TMADH⅐ETF complex was then plotted versus the number of electrons added. The experiment was repeated three times, and the total number of electrons taken up obtained were averaged. Since the titration of FMN was performed after the titration of the protein solution, the values obtained are expected to slightly underestimate the electron uptake of the system due to slow oxidation of the dithionite stock solution.

RESULTS
Spectral Changes Observed for the Reaction of the TMADH⅐ETF Complex with Trimethylamine-The spectral changes associated with the reaction of the TMADH⅐ETF complex with TMA are shown in Fig. 1. 2 The spectral change observed within 10 s after addition of TMA to the complex (Fig.  1B) is consistent with reduction of the complex by two equivalents of TMA to give the four-electron reduced state, with three electrons required for full reduction of TMADH and one for reducing ETF to the semiquinone form. Subsequently, however, an additional spectral change is observed. This slower spectral change is shown in Fig. 1C and has features, in particular the substantial absorbance decrease at 370 nm reflecting loss of the anionic semiquinone, that suggest further reduction of ETF semiquinone to the hydroquinone state has occurred.
Dithionite Titration of the TMADH⅐ETF Complex-To investigate whether ETF can indeed be reduced fully to the flavin hydroquinone within the TMADH⅐ETF complex, a quantitative titration with sodium dithionite was performed to determine the total number of reducing equivalents taken up by the protein complex. The absorption spectra of the protein complex in the course of the titration are shown in Fig. 2A. A plot of the absorbance change at 370 nm (following the accumulation and subsequent decay of the anionic semiquinone of both TMADH and ETF) versus the number of reducing equivalents added is shown in Fig. 2B, where it is evident that approximately five electrons are required for full reduction of the complex and that at completion of the titration the absorption at 370 nm, which accumulates transiently in the course of the titration, has been lost, undoubtedly reflecting reduction of the ETF semiquinone on to the hydroquinone. The experiment was performed in triplicate, and the average electron uptake of the complex found to be 4.7 Ϯ 0.12 e Ϫ . Assuming a K d for binding of the oxidized forms of the two proteins comparable to the value of 10 M determined kinetically for complex formation between reduced TMADH and oxidized ETF, it is estimated that about 73% of the proteins would form complex under the present experimental conditions. Therefore, a value of 4.7 electrons actually agrees very well with the fact that not all of the protein molecules have formed complex and only those in the complex were able to take up an additional electron. Control experiments were also performed to ensure that the FAD cofactor did not dissociate from ETF in the course of dithionite titration, and this was found not to be the case. 3 The results of the dithionite titration clearly demonstrate that the TMADH⅐ETF complex can indeed take up a total of five electrons and that ETF can be reduced fully to FAD hydroquinone when it is in complex with TMADH.
Reaction of Three-electron Reduced TMADH (TMADH 3eϪ ) with ETF Semiquinone (ETF sq )-To investigate whether full reduction of ETF within the TMADH⅐ETF complex occurs by 2 This reaction was necessarily performed under aerobic conditions, as the 1-mm light path cuvette that had to be used (owing to the high concentrations of proteins used) had an open top and it was not technically feasible to make it anaerobic. We note that both reduced TMADH and ETF are unusually stable to air-reoxidation, however, with half-lives for the reaction of approximately 1 h. In particular, it has been shown previously that the reaction of TMADH with TMA (in the absence of ETF) exhibits identical kinetics under aerobic and anaerobic conditions (24,25). For these reasons, the effect of O 2 on the present reaction is expected to be negligible. That this is the case is evident in the sustained reduction of the enzyme that is observed over the duration of the present experiments. All spectral changes that are observed, even those on the slowest (seconds) time scale, are associated with reduction of the enzyme-ETF complex, and not its reoxidation. 3 After the dithionite titration was complete, the reduced sample was loaded to a small Sephadex G-25 column to determine whether a fraction of free FAD was present. If FAD had dissociated from the protein, it would be rapidly reoxidized by O 2 during gel chromatography and a bright yellow band would appear. The column was also illuminated with UV light to detect any trace amount of FAD, which is highly fluorescent. No yellow fraction or fluorescence was observed for the dithionite-reduced protein sample during the whole process of chromatography, indicating that the FAD cofactor did not dissociate from ETF in the course of dithionite titration. Ϫ (oxidized complex)). C, spectral change observed in the 10 min after adding TMA to the protein complex ((TMA-reduced complex, 10 min after adding TMA) Ϫ (TMA-reduced complex right after mixing)). electron transfer from TMADH rather than direct reduction by sodium dithionite, the following experiment was performed. Equal volumes of 70 M TMADH 3eϪ and 70 M ETF sq were placed into separate compartments of an anaerobic split-cell cuvette and the spectrum recorded. The two solutions were then mixed thoroughly, and the spectrum of the sample was measured every 20 s for 15 min (the reaction was found to go to completion in approximately 10 min). The spectra of the solution before mixing and upon completion of the reaction are shown in Fig. 3A, and the ((15 min after mixing) Ϫ (before mixing)) difference spectrum in Fig. 3B. Since the spectra for TMADH 3eϪ , TMADH 2eϪ , ETF sq , and ETF hq are known, the expected spectral change associated with transfer of one electron from TMADH 3eϪ to ETF sq to give TMADH 2eϪ (containing FMN hq and oxidized 4Fe/4S center) 4 and ETF hq can be calculated, and is shown in Fig. 3C. From a comparison of panels B and C, it is evident that the experimentally obtained difference spectrum agrees well with that calculated from the known spectral properties of TMADH and ETF, 5 indicating that electron transfer from TMADH 3eϪ (presumably via its 4Fe/4S center; see below) to ETF sq was achieved. From the magnitude of the spectral change at 370 nm, it is estimated that electron transfer went to only 30% completion under the present experimental conditions, reflecting an internal equilibrium of the electron distribution between TMADH 3eϪ ⅐ETF sq and TMADH 2eϪ ⅐ETF hq .
Reaction of Reduced Phenylhydrazine-inactivated TMADH (PHZ-TMADH 1eϪ ) with ETF Semiquinone (ETF sq )-It is known that in the course of turnover, reducing equivalents are transferred from TMADH to ETF exclusively via the 4Fe/4S site of the former to give the ETF anionic semiquinone (20). To confirm that this is also the case for full reduction of ETF to the hydroquinone level, phenylhydrazine-inactivated TMADH was used in an experiment analogous to that described above with TMADH 3eϪ . Reaction with phenylhydrazine results in covalent modification of the FMN cofactor of TMADH at the C-4a position, rendering it redox-inert, so that only the 4Fe/4S center 4 At pH 7.0, the electron distribution in two-electron reduced TMADH favors formation of FMN hq and oxidized 4Fe/4S center (31). Therefore, the 4Fe/4S center remains predominantly oxidized after transferring one electron to the FAD of ETF since electron transfer from FMN hq to the 4Fe/4S center is under prototropic control and essentially prevented at pH 7.0. 5 The slight difference seen between the observed and calculated difference spectra is most likely a result of the minor spectral perturbation associated with binding of ETF to TMADH, which is known to occur when the oxidized form of the two proteins bind to each other. FIG. 3. Spectral change associated with the reaction of TMADH 3e؊ with ETF sq . A, absorption spectrum of TMADH 3eϪ and ETF sq in separate compartments of a split cell cuvette (solid line), and spectrum of the sample after the two solutions were mixed (⅐⅐⅐). B, difference absorption spectrum observed with the reaction of TMADH 3eϪ with ETF sq ((after mixing) Ϫ (before mixing)). C, calculated spectral change associated with one-electron transfer from TMADH 3eϪ to ETF sq to give TMADH 2eϪ (containing FMN hq and oxidized 4Fe/4S center) and ETF hq . The concentrations of TMADH 3eϪ and ETF sq before mixing are 70 M for each protein.
remains redox-active (23,27). The spectral change observed upon mixing PHZ-TAMDH 1eϪ with ETF sq is shown in Fig. 4. The spectra of the protein solution before mixing and 15 min after mixing are shown in Fig. 4A, and the ((15 min after mixing) Ϫ (before mixing)) difference spectrum in Fig. 4B. The expected spectral change for electron transfer from PHZ-TMADH 1eϪ to ETF sq forming PHZ-TMADH ox and ETF hq was also calculated and is shown in Fig. 4C. It is evident that the experimentally obtained difference spectrum is again very similar to the calculated one, demonstrating that phenylhydrazine-treated TMADH is still capable of transferring reducing equivalents to further reduce ETF from the semiquinone to hydroquinone. 5 Based on the observed spectral change at 370 nm, it is estimated that electron transfer has occurred to the extent of 56% in the course of the reaction. As in the case of the reaction with native enzyme, this presumably reflects an internal equilibrium between PHZ-TMADH 1eϪ ⅐ETF sq and PHZ-TMADH ox ⅐ETF hq .
Binding of Oxidized ETF to Oxidized TMADH-Finally, in order to obtain direct evidence for formation of a complex between TMADH and ETF, a split cell experiment was performed with the oxidized forms of both proteins to determine whether a discernible spectral change is observed. A substantial spectral change is indeed seen for the binding of oxidized ETF to TMADH, as shown in Fig. 5, characterized by two prominent positive features at 490 and 400 nm, and a negative feature centered at 440 nm. Again assuming a K d for binding oxidized TMADH and ETF comparable to the kinetically determined K d of 10 M for reduced TMADH and oxidized ETF (20), it is estimated that 61% of each of the two proteins are complexed under the present experimental conditions (the concentrations of TMADH and ETF after mixing were each 40 M). Taking into account the complex concentration thus calculated (ϳ24 M), an extinction change associated with complex formation of approximately 2 mM Ϫ1 cm Ϫ1 at 490 nm can be obtained, as indicated in Fig. 5B. 6 The form of the spectral change is 6 Although falling in the range of 2-4 mM Ϫ1 cm Ϫ1 seen with other FIG. 4. Spectral change associated with the reaction of reduced phenylhydrazine-inactivated TMADH with ETF sq . A, absorption spectrum of reduced phenylhydrazine-inactivated TMADH (PHZ-TMADH 1eϪ ) and ETF sq in separate compartments of a split-cell cuvette (solid line), and spectrum of the sample after the two solutions were mixed (⅐⅐⅐). B, difference absorption spectrum observed with the reaction of PHZ-TMADH 1eϪ with ETF sq ((after mixing) Ϫ (before mixing)). C, calculated spectral change associated with one-electron transfer from PHZ-TMADH 1eϪ to ETF sq to give PHZ-TMADH ox and ETF hq . The concentrations of PHZ-TMADH 1eϪ and ETF sq before mixing are 70 M for each protein. consistent with taking a normal unmodified flavin from a more hydrophilic environment into a more hydrophobic one (32)(33)(34)(35) and must therefore be due to a perturbation of the environment of the FAD in ETF, rather than the 6-S-cysteinyl FMN of TMADH. DISCUSSION In the present study, we have demonstrated that full reduction of W 3 A 1 ETF can be readily achieved both enzymatically (with excess substrate) and chemically (by sodium dithionite) within the TMADH⅐ETF protein complex. Transfer of a second reducing equivalent to the ETF semiquinone to give the hydroquinone is mediated by TMADH, as demonstrated by the occurrence of electron transfer upon mixing either TMADH 3eϪ or PHZ-TMADH 1eϪ with ETF sq under anaerobic conditions. Previous studies have shown that ETF receives a first reducing equivalent from TMADH exclusively via the 4Fe/4S center in the course of the oxidative halfreaction (20). The fact that reduced phenylhydrazine-inactivated TMADH is also able to transfer an electron to ETF semiquinone for the formation of hydroquinone indicates that the second reducing equivalent is also transferred via the 4Fe/4S center of TMADH, as expected.
The reduction potentials of the redox-active centers in TMADH and ETF have been determined previously. At pH 7.0, the FMN/FMN sq , FMN sq /FMN hq , and FeS ox /FeS red couples of TMADH have potentials of 44, 36, and 110 mV, respectively (22), while the potentials for the FAD/FAD sq and FAD sq /FAD hq couples of ETF are 196 and Ϫ197 mV (21). The high potential for the FAD/FAD sq couple (196 mV) is consistent with its physiological role in receiving electrons from the 4Fe/4S center of TMADH (110 mV), but that for the FAD sq /FAD hq couple in ETF (Ϫ197 mV) is substantially lower than those of the centers in TMADH, and further reduction is therefore thermodynamically unfavorable. Nevertheless, the results presented here clearly demonstrate that ETF is able to receive a second reducing equivalent from the 4Fe/4S center of TMADH and become fully reduced when it is in complex with TMADH. This indicates that the relative reduction potentials of the cofactors must be perturbed upon formation of the TMADH⅐ETF complex.
From the magnitude of the spectral change associated with the reaction of fully reduced TMADH with ETF semiquinone, it is estimated that electron transfer has taken place in about 30% of the TMADH 3eϪ ⅐ETF sq complex to give TMADH 2eϪ ⅐ETF hq . Given the extent of electron transfer that is observed, the reduction potential of the FAD sq /FAD hq couple of ETF within the complex must be shifted upward by on the order of 200 mV (from that seen in free solution) to a value in the range of 0 mV. So large a change in reduction potential must reflect a substantial change in the environment of the FAD of ETF upon binding to TMADH. On the other hand, electron transfer is found to occur in about 56% of the PHZ-TMADH 1eϪ ⅐ETF sq protein complex. Since these percentages reflect the internal equilibrium of electron distribution between the cofactors, the result indicates that the reduction potentials for the cofactors involved (specifically the 4Fe/4S center and FAD cofactor) may be affected to a certain extent by covalent modification of the FMN cofactor in TMADH. According to the Nernst equation, such a difference in equilibrium or electron distribution (30% versus 56%) reflects a moderate ϳ20-mV difference in relative reduction potentials of the FeS ox / FeS red and FAD sq /FAD hq redox couples.
Bearing in mind the uncertainties inherent in the above argument, a set of reduction potentials for the TMADH⅐ETF complex predicted from the above arguments can be given as: FMN/FMN sq , 44 mV; FMN sq /FMN hq , 36 mV; FeS ox /FeS red 110 mV; FAD/FAD sq , 196 mV; FAD sq /FAD hq , 0 mV. Given this set of potentials, the first reducing equivalent is expected to yield principally the ETF (anionic) semiquinone, with a concomitant increase in absorbance at 370 nm. A second equivalent should go to reduce the Fe/S center of TMADH, although some FMN semiquinone is expected to accumulate. The significant further increase in absorption at 370 nm that is observed experimentally ( Fig. 2) appears greater than might have been expected at this stage of the titration, but it is important to recognize that addition of two reducing equivalents is not likely to generate a homogeneous population of two-electron reduced complex, but rather a distribution consisting principally of one-, two-, and three-electron reduced complex, as has been found to be the case in other complex redox-active systems (see, e.g. Ref. 36). By addition of the third reducing equivalent, however, the absorption increase at 370 nm seen in the course of the reductive titration is at least qualitatively consistent with a system containing ETF essentially completely as the semiquinone, and the FMN of TMADH Ͼ33% in the semiquinone form also, with the Fe/S center of TMADH for the most part reduced. Subsequent bleaching of the absorbance at 370 nm as the titration goes to completion clearly indicates the eventual reduction of both the FAD of ETF and the FMN of TMADH to the level of the hydroquinone. It is to be emphasized that this analysis must be regarded as only qualitative, and it is possible that the extent of the transient increase in absorbance at 370 nm observed experimentally reflects some perturbation of the TMADH flavin potentials in addition to that discussed for the ETF SQ/HQ couple, giving rise to a somewhat greater thermodynamic stabilization of the semiquinone of this site than expected on the basis of the reduction potentials for the free enzyme. Future work involving the explicit determination of the reduction potentials of the several sites within the TMADH⅐ETF complex will address this explicitly. The somewhat qualitative nature of this argument concerning the behavior of the complex in the course of a reductive titration notwithstanding, however, we emphasize that the observation of substantial electron transfer from reduced TMADH to the ETF semiquinone (Figs. 3 and 4) is most readily accounted for by invoking an increase in potential for the ETF SQ/HQ couple of on the order of 200 mV.
A three-dimensional structural model for W 3 A 1 ETF has been constructed using homology modeling techniques (26), based on the crystal structure of human ETF (9). On the basis of this model and the x-ray crystal structure of TMADH (37), it is evident that an electron transfer-efficient complex cannot be formed by the two proteins without a significant structural reorganization. In particular, both the regions surrounding Tyr-442 in TMADH and the FAD of ETF are concave and not physically complementary. It thus has been suggested that ETF undergoes a substantial conformational change upon flavoproteins (32)(33)(34)(35), this extinction coefficient must be regarded with some caution, based as it is on the calculated concentration of complex at 40 M each of TMADH and ETF using a kinetically determined K d of 10 M for reduced TMADH and oxidized ETF. In principle it is possible to ascertain the K d for the oxidized forms of both proteins in a true equilibrium experiment from the extent of the spectral change shown in Fig. 5 at other concentrations of TMADH and ETF. As 40 M each of TMADH and ETF is the highest concentration at which we can work in the present experiments, given the constraints of Beer's law, we also performed experiments of the type shown in Fig. 5 at lower concentrations as part of the present study. Unfortunately, while the small spectral change observed was generally consistent with that expected for a K d of 10 M, the signal-to-noise was extremely poor, presumably due to the combined factors of an intrinsically smaller spectral change and a large background absorbance in the 400 -500 nm region. For this reason, we are unable at present to say more than that our data are consistent with a K d of 10 M and an extinction change of 2 mM Ϫ1 cm Ϫ1 . binding to TMADH, with domains I and III rotating ϳ50°with respect to domain II relative to their initial positions in the structural model (26,38). Within the protein complex, the conformation adopted by ETF is proposed to be conducive to electron transfer, with the FAD cofactor properly oriented for efficient electron transfer from the 4Fe/4S center of TMADH. In the context of such a structural rearrangement, it is not surprising that the reduction potential of ETF in the electron transfer-active form is perturbed. In addition, the FAD of ETF goes from being exceptionally exposed to solvent to a generally more hydrophobic environment, consistent with the spectral change observed upon binding to TMADH. Our results are thus entirely consistent with the idea that a conformational change in ETF occurs upon binding to TMADH and perturbs the oxidation-reduction properties of ETF, thus facilitating the transfer of a second electron into ETF to allow full reduction of the protein.
In the present work, we have demonstrated that W 3 A 1 ETF can be reduced fully to the flavin hydroquinone form both enzymatically and chemically within the TMADH⅐ETF complex. This reaction is considerably more facile than is the case with ETF in free solution, taking place on a time scale of minutes rather than hours. It is important to bear in mind that full reduction of the TMADH⅐ETF complex requires five electrons, and when substrate is used, an intercomplex dismutation must take place between two equivalents of [TMADH⅐ETF] 4eϪ to give one equivalent each of [TMADH⅐ETF] 5eϪ and [TMADH⅐ETF] 3eϪ ; this latter species can then react with a final equivalent of TMA to give complete reduction of the system. Given that this dismutation is ratelimiting for full reduction, it is not surprising that the accumulation of fully reduced ETF is relatively slow. On the other hand, the spectral changes seen upon mixing TMADH 3eϪ or PHZ-TMADH 1eϪ with ETF sq also occur on a time scale of minutes, indicating that reduction to the hydroquinone is not particularly fast. It is thus not clear that the hydroquinone form of ETF accumulates to a significant extent under normal conditions, given this slow rate of the reduction. The significance of the present work, however, is that it unequivocally demonstrates that the oxidation-reduction properties of the cofactors have been altered upon formation of the TMADH⅐ETF protein complex (especially that of the FAD sq /FAD hq couple in ETF), consistent with the proposed structural changes of ETF during complex assembly. Further, the spectral change seen here that is associated with the binding of the two proteins demonstrates unequivocally that a complex does form and provides a new and convenient experimental probe whereby the protein protein interaction can be monitored.