Probing the dynamic interface between trimethylamine dehydrogenase (TMADH) and electron transferring flavoprotein (ETF) in the TMADH-2ETF complex: role of the Arg-alpha237 (ETF) and Tyr-442 (TMADH) residue pair

We have used multiple solution state techniques and crystallographic analysis to investigate the importance of a putative transient interaction formed between Arg-alpha237 in electron transferring flavoprotein (ETF) and Tyr-442 in trimethylamine dehydrogenase (TMADH) in complex assembly, electron transfer, and structural imprinting of ETF by TMADH. We have isolated four mutant forms of ETF altered in the identity of the residue at position 237 (alphaR237A, alphaR237K, alphaR237C, and alphaR237E) and with each form studied electron transfer from TMADH to ETF, investigated the reduction potentials of the bound ETF cofactor, and analyzed complex formation. We show that mutation of Arg-alpha237 substantially destabilizes the semiquinone couple of the bound FAD and impedes electron transfer from TMADH to ETF. Crystallographic structures of the mutant ETF proteins indicate that mutation does not perturb the overall structure of ETF, but leads to disruption of an electrostatic network at an ETF domain boundary that likely affects the dynamic properties of ETF in the crystal and in solution. We show that Arg-alpha237 is required for TMADH to structurally imprint the as-purified semiquinone form of wild-type ETF and that the ability of TMADH to facilitate this structural reorganization is lost following (i) redox cycling of ETF, or simple conversion to the oxidized form, and (ii) mutagenesis of Arg-alpha237. We discuss this result in light of recent apparent conflict in the literature relating to the structural imprinting of wild-type ETF. Our studies support a mechanism of electron transfer by conformational sampling as advanced from our previous analysis of the crystal structure of the TMADH-2ETF complex [Leys, D. , Basran, J. , Sutcliffe, M. J., and Scrutton, N. S. (2003) Nature Struct. Biol. 10, 219-225] and point to a key role for the Tyr-442 (TMADH) and Arg-alpha237 (ETF) residue pair in transiently stabilizing productive electron transfer configurations. Our work also points to the importance of Arg-alpha237 in controlling the thermodynamics of electron transfer, the dynamics of ETF, and the protection of reducing equivalents following disassembly of the TMADH-2ETF complex.     

Flavin radicals, conformational sampling and robust design principles in interprotein electron transfer: the trimethylamine dehydrogenase-electron-transferring flavoprotein complex

TMADH (trimethylamine dehydrogenase) is a complex iron-sulphur flavoprotein that forms a soluble electron-transfer complex with ETF (electron-transferring flavoprotein). The mechanism of electron transfer between TMADH and ETF has been studied using stopped-flow kinetic and mutagenesis methods, and more recently by X-ray crystallography. Potentiometric methods have also been used to identify key residues involved in the stabilization of the flavin radical semiquinone species in ETF. These studies have demonstrated a key role for 'conformational sampling' in the electron-transfer complex, facilitated by two-site contact of ETF with TMADH. Exploration of three-dimensional space in the complex allows the FAD of ETF to find conformations compatible with enhanced electronic coupling with the 4Fe-4S centre of TMADH. This mechanism of electron transfer provides for a more robust and accessible design principle for interprotein electron transfer compared with simpler models that invoke the collision of redox partners followed by electron transfer. The structure of the TMADH-ETF complex confirms the role of key residues in electron transfer and molecular assembly, originally suggested from detailed kinetic studies in wild-type and mutant complexes, and from molecular modelling.     

Electron transfer and conformational change in complexes of trimethylamine dehydrogenase and electron transferring flavoprotein.

The trimethylamine dehydrogenase-electron transferring flavoprotein (TMADH.ETF) electron transfer complex has been studied by fluorescence and absorption spectroscopies. These studies indicate that a series of conformational changes occur during the assembly of the TMADH.ETF electron transfer complex and that the kinetics of assembly observed with mutant TMADH (Y442F/L/G) or ETF (alpha R237A) complexes are much slower than are the corresponding rates of electron transfer in these complexes. This suggests that electron transfer does not occur in the thermodynamically most favorable state (which takes too long to form), but that one or more metastable states (which are formed more rapidly) are competent in transferring electrons from TMADH to ETF. Additionally, fluorescence spectroscopy studies of the TMADH.ETF complex indicate that ETF undergoes a stable conformational change (termed structural imprinting) when it interacts transiently with TMADH to form a second, distinct, structural form. The mutant complexes compromise imprinting of ETF, indicating a dependence on the native interactions present in the wild-type complex. The imprinted form of semiquinone ETF exhibits an enhanced rate of electron transfer to the artificial electron acceptor, ferricenium. Overall molecular conformations as probed by small-angle x-ray scattering studies are indistinguishable for imprinted and non-imprinted ETF, suggesting that changes in structure likely involve confined reorganizations within the vicinity of the FAD. Our results indicate a series of conformational events occur during the assembly of the TMADH.ETF electron transfer complex, and that the properties of electron transfer proteins can be affected lastingly by transient interaction with their physiological redox partners. This may have significant implications for our understanding of biological electron transfer reactions in vivo, because ETF encounters TMADH at all times in the cell. Our studies suggest that caution needs to be exercised in extrapolating the properties of in vitro interprotein electron transfer reactions to those occurring in vivo.     

Redox cycles in trimethylamine dehydrogenase and mechanism of substrate inhibition.

The steady-state reaction of trimethylamine dehydrogenase (TMADH) with the artificial electron acceptor ferricenium hexafluorophosphate (Fc(+)) has been studied by stopped-flow spectroscopy, with particular reference to the mechanism of inhibition by trimethylamine (TMA). Previous studies have suggested that the presence of alternate redox cycles is responsible for the inhibition of activity seen in the high-substrate regime. Here, we demonstrate that partitioning between these redox cycles (termed the 0/2 and 1/3 cycles on the basis of the number of reducing equivalents present in the oxidized/reduced enzyme encountered in each cycle) is dependent on both TMA and electron acceptor concentration. The use of Fc(+) as electron acceptor has enabled a study of the major redox forms of TMADH present during steady-state turnover at different concentrations of substrate. Reduction of Fc(+) is found to occur via the 4Fe-4S center of TMADH and not the 6-S-cysteinyl flavin mononucleotide: the direction of electron flow is thus analogous to the route of electron transfer to the physiological electron acceptor, an electron-transferring flavoprotein (ETF). In steady-state reactions with Fc(+) as electron acceptor, partitioning between the 0/2 and 1/3 redox cycles is dependent on the concentration of the electron acceptor. In the high-concentration regime, inhibition is less pronounced, consistent with the predicted effects on the proposed branching kinetic scheme. Photodiode array analysis of the absorption spectrum of TMADH during steady-state turnover at high TMA concentrations reveals that one-electron reduced TMADH-possessing the anionic flavin semiquinone-is the predominant species. Conversely, at low concentrations of TMA, the enzyme is predominantly in the oxidized form during steady-state turnover. The data, together with evidence derived from enzyme-monitored turnover experiments performed at different concentrations of TMA, establish the operation of the branched kinetic scheme in steady-state reactions. With dimethylbutylamine (DMButA) as substrate, the partitioning between the 0/2 and 1/3 redox cycles is poised more toward the 0/2 cycle at all DMButA concentrations studied-an observation that is consistent with the inability of DMButA to act as an effective inhibitor of TMADH.     

The iron-sulfur cluster of electron transfer flavoprotein-ubiquinone oxidoreductase is the electron acceptor for electron transfer flavoprotein

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) accepts electrons from electron transfer flavoprotein (ETF) and reduces ubiquinone from the ubiquinone pool. It contains one [4Fe-4S] (2+,1+) and one FAD, which are diamagnetic in the isolated oxidized enzyme and can be reduced to paramagnetic forms by enzymatic donors or dithionite. In the porcine protein, threonine 367 is hydrogen bonded to N1 and O2 of the flavin ring of the FAD. The analogous site in Rhodobacter sphaeroides ETF-QO is asparagine 338. Mutations N338T and N338A were introduced into the R. sphaeroides protein by site-directed mutagenesis to determine the impact of hydrogen bonding at this site on redox potentials and activity. The mutations did not alter the optical spectra, EPR g-values, spin-lattice relaxation rates, or the [4Fe-4S] (2+,1+) to FAD point-dipole interspin distances. The mutations had no impact on the reduction potential for the iron-sulfur cluster, which was monitored by changes in the continuous wave EPR signals of the [4Fe-4S] (+) at 15 K. For the FAD semiquinone, significantly different potentials were obtained by monitoring the titration at 100 or 293 K. Based on spectra at 293 K the N338T mutation shifted the first and second midpoint potentials for the FAD from +47 and -30 mV for wild type to -11 and -19 mV, respectively. The N338A mutation decreased the potentials to -37 and -49 mV. Lowering the midpoint potentials resulted in a decrease in the quinone reductase activity and negligible impact on disproportionation of ETF 1e (-) catalyzed by ETF-QO. These observations indicate that the FAD is involved in electron transfer to ubiquinone but not in electron transfer from ETF to ETF-QO. Therefore, the iron-sulfur cluster is the immediate acceptor from ETF.     

The interaction of trimethylamine dehydrogenase and electron-transferring flavoprotein

The interaction between the physiological electron transfer partners trimethylamine dehydrogenase (TMADH) and electron-transferring flavoprotein (ETF) from Methylophilus methylotrophus has been examined with particular regard to the proposal that the former protein "imprints" a conformational change on the latter. The results indicate that the absorbance change previously attributed to changes in the environment of the FAD of ETF upon binding to TMADH is instead caused by electron transfer from partially reduced, as-isolated TMADH to ETF. Prior treatment of the as-isolated enzyme with the oxidant ferricenium essentially abolishes the observed spectral change. Further, when the semiquinone form of ETF is used instead of the oxidized form, the mirror image of the spectral change seen with as-isolated TMADH and oxidized ETF is observed. This is attributable to a small amount of electron transfer in the reverse of the physiological direction. Kinetic determination of the dissociation constant and limiting rate constant for electron transfer within the complex of (reduced) TMADH with (oxidized) ETF is reconfirmed and discussed in the context of a recently proposed model for the interaction between the two proteins that involves "structural imprinting" of ETF.     

Thermodynamic basis of electron transfer in dihydroorotate dehydrogenase B from Lactococcus lactis: analysis by potentiometry, EPR spectroscopy, and ENDOR spectroscopy

Dihydroorotate dehydrogenase B (DHODB) is a complex iron-sulfur flavoprotein that catalyzes the conversion of dihydroorotate to orotate and the reduction of NAD(+). The enzyme is a dimer of heterodimers containing an FMN, an FAD, and a 2Fe-2S center. UV-visible, EPR, and ENDOR spectroscopies have been used to determine the reduction potentials of the flavins and the 2Fe-2S center and to characterize radicals and their interactions. Reductive titration using dithionite indicates a five-electron capacity for DHODB. The midpoint reduction potential of the 2Fe-2S center (-212 +/- 3 mV) was determined from analysis of absorption data at 540 nm, where absorption contributions from the two flavins are small. The midpoint reduction potentials of the oxidized/semiquinone (E(1)) and semiquinone/hydroquinone (E(2)) couples for the FMN (E(1) = -301 +/- 6 mV; E(2) = -252 +/- 8 mV) and FAD (E(1) = -312 +/- 6 mV; E(2) = -297 +/- 5 mV) were determined from analysis of spectral changes at 630 nm. Corresponding values for the midpoint reduction potentials for FMN (E(1) = -298 +/- 4 mV; E(2) = -259 +/- 5 mV) in the isolated catalytic subunit (subunit D, which lacks the 2Fe-2S center and FAD) are consistent with the values determined for the FMN couples in DHODB. During reductive titration of DHODB, small amounts of the neutral blue semiquinone are observed at approximately 630 nm, consistent with the measured midpoint reduction potentials of the flavins. An ENDOR spectrum of substrate-reduced DHODB identifies hyperfine couplings to proton nuclei similar to those recorded for the blue semiquinone of free flavins in aqueous solution, thus confirming the presence of this species in DHODB. Spectral features observed during EPR spectroscopy of dithionite-reduced DHODB are consistent with the midpoint reduction potentials determined using UV-visible spectroscopy and further identify an unusual EPR signal with very small rhombic anisotropy and g values of 2.02, 1.99, and 1.96. This unusual signal is assigned to the formation of a spin interacting state between the FMN semiquinone species and the reduced 2Fe-2S center. Reduction of DHODB using an excess of NADH or dihydroorotate produces EPR spectra that are distinct from those produced by dithionite. From potentiometric studies, the reduction of the 2Fe-2S center and the reduction of the FMN occur concomitantly. The study provides a detailed thermodynamic framework for electron transfer in this complex iron-sulfur flavoprotein.     

Lys-D48 is required for charge stabilization, rapid flavin reduction, and internal electron transfer in the catalytic cycle of dihydroorotate dehydrogenase B of Lactococcus lactis

Dihydroorotate dehydrogenase B (DHODB) catalyzes the oxidation of dihydroorotate (DHO) to orotate and is found in the pyrimidine biosynthetic pathway. The Lactococcus lactis enzyme is a dimer of heterodimers containing FMN, FAD, and a 2Fe-2S center. Lys-D48 is found in the catalytic subunit and its side-chain adopts different positions, influenced by ligand binding. Based on crystal structures of DHODB in the presence and absence of orotate, we hypothesized that Lys-D48 has a role in facilitating electron transfer in DHODB, specifically in stabilizing negative charge in the reduced FMN isoalloxazine ring. We show that mutagenesis of Lys-D48 to an alanine, arginine, glutamine, or glutamate residue (mutants K38A, K48R, K48Q, and K48E) impairs catalytic turnover substantially (approximately 50-500-fold reduction in turnover number). Stopped-flow studies demonstrate that loss of catalytic activity is attributed to poor rates of FMN reduction by substrate. Mutation also impairs electron transfer from the 2Fe-2S center to FMN. Addition of methylamine leads to partial rescue of flavin reduction activity. Nicotinamide coenzyme oxidation and reduction at the distal FAD site is unaffected by the mutations. Formation of the spin-interacting state between the FMN semiquinone-reduced 2Fe-2S centers observed in wild-type enzyme is retained in the mutant proteins, consistent with there being little perturbation of the superexchange paths that contribute to the efficiency of electron transfer between these cofactors. Our data suggest a key charge-stabilizing role for Lys-D48 during reduction of FMN by dihydroorotate, or by electron transfer from the 2Fe-2S center, and establish a common mechanism of FMN reduction in the single FMN-containing A-type and the complex multicenter B-type DHOD enzymes.     

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.     

The crystal structure and reaction mechanism of Escherichia coli 2,4-dienoyl-CoA reductase

Escherichia coli 2,4-dienoyl-CoA reductase is an iron-sulfur flavoenzyme required for the metabolism of unsaturated fatty acids with double bonds at even carbon positions. The enzyme contains FMN, FAD, and a 4Fe-4S cluster and exhibits sequence homology to another iron-sulfur flavoprotein, trimethylamine dehydrogenase. It also requires NADPH as an electron source, resulting in reduction of the C4-C5 double bond of the acyl chain of the CoA thioester substrate. The structure presented here of a ternary complex of E. coli 2,4-dienoyl-CoA reductase with NADP+ and a fatty acyl-CoA substrate reveals a possible mechanism for substrate reduction and provides details of a plausible electron transfer mechanism involving both flavins and the iron-sulfur cluster. The reaction is initiated by hydride transfer from NADPH to FAD, which in turn transfers electrons, one at a time, to FMN via the 4Fe-4S cluster. In the final stages of the reaction, the fully reduced FMN provides a hydride ion to the C5 atom of substrate, and Tyr-166 and His-252 are proposed to form a catalytic dyad that protonates the C4 atom of the substrate and complete the reaction. Inspection of the substrate binding pocket explains the relative promiscuity of the enzyme, catalyzing reduction of both 2-trans,4-cis- and 2-trans,4-trans-dienoyl-CoA thioesters.     

Determination of the midpoint potential of the FAD and FMN flavin cofactors and of the 3Fe-4S cluster of glutamate synthase

Glutamate synthase is a complex iron-sulfur flavoprotein that catalyzes the reductive transfer of the L-glutamine amide group to C(2) of 2-oxoglutarate, forming two molecules of L-glutamate. The bacterial enzyme is an alphabeta protomer, which contains one FAD (on the beta subunit, approximately 50 kDa), one FMN (on the alpha subunit, approximately 150 kDa), and three different Fe-S clusters (one 3Fe-4S center on the alpha subunit and two 4Fe-4S clusters at an unknown location). To address the problem of the intramolecular electron pathway, we have measured the midpoint potential values of the flavin cofactors and of the 3Fe-4S cluster of glutamate synthase in the isolated alpha and beta subunits and in the alphabeta holoenzyme. No detectable amounts of flavin semiquinones were observed during reductive titrations of the enzyme, indicating that the midpoint potential value of each flavin(ox)/flavin(sq) couple is, in all cases, significantly more negative than that of the corresponding flavin(sq)/flavin(hq) couple. Association of the two subunits to form the alphabeta protomer does not alter significantly the midpoint potential value of the FMN cofactor and of the 3Fe-4S cluster (approximately -240 and -270 mV, respectively), but it makes that of FAD some 40 mV less negative (approximately -340 mV for the beta subunit and -300 mV for FAD bound to the holoenzyme). Binding of the nonreducible NADP(+) analogue, 3-aminopyridine adenine dinucleotide phosphate, made the measured midpoint potential value of the FAD cofactor approximately 30-40 mV less negative in the isolated beta subunit, but had no effect on the redox properties of the alphabeta holoenzyme. This result correlates with the formation of a stable charge-transfer complex between the reduced flavin and the oxidized pyridine nucleotide in the isolated beta subunit, but not in the alphabeta holoenzyme. Binding of L-methionine sulfone, a glutamine analogue, had no significant effect on the redox properties of the enzyme cofactors. On the contrary, 2-oxoglutarate made the measured midpoint potential value of the 3Fe-4S cluster approximately 20 mV more negative in the isolated alpha subunit, but up to 100 mV less negative in the alphabeta holoenzyme as compared to the values of the corresponding free enzyme forms. These findings are consistent with electron transfer from the entry site (FAD) to the exit site (FMN) through the 3Fe-4S center of the enzyme and the involvement of at least one of the two low-potential 4Fe-4S centers, which are present in the glutamate synthase holoenzyme, but not in the isolated subunits. Furthermore, the data demonstrate a specific role of 2-oxoglutarate in promoting electron transfer from FAD to the 3Fe-4S cluster of the glutamate synthase holoenzyme. The modulatory role of 2-oxoglutarate is indeed consistent with the recently determined three-dimensional structure of the glutamate synthase alpha subunit, in which several polypeptide stretches are suitably positioned to mediate communication between substrate binding sites and the enzyme redox centers (FMN and the 3Fe-4S cluster) to tightly control and coordinate the individual reaction steps [Binda, C., et al. (2000) Structure 8, 1299-1308].     

The role of Tyr-169 of trimethylamine dehydrogenase in substrate oxidation and magnetic interaction between FMN cofactor and the 4Fe/4S center

Tyr-169 in trimethylamine dehydrogenase is one component of a triad also comprising residues His-172 and Asp-267. Its role in catalysis and in mediating the magnetic interaction between FMN cofactor and the 4Fe/4S center have been investigated by stopped-flow and EPR spectroscopy of a Tyr-169 to Phe (Y169F) mutant of the enzyme. Tyr-169 is shown to play an important role in catalysis (mutation to phenylalanine reduces the limiting rate constant for bleaching of the active site flavin by about 100-fold) but does not serve as a general base in the course of catalysis. In addition, we are able to resolve two kinetically influential ionizations involved in both the reaction of free enzyme with free substrate (as reflected in klim/Kd), and in the breakdown of the Eox.S complex (as reflected in klim). In EPR studies of the Y169F mutant, it is found that the ability of the Y169F enzyme to form the spin-interacting state between flavin semiquinone and reduced 4Fe/4S center characteristic of wild-type enzyme is significantly compromised. The present results are consistent with Tyr-169 representing the ionizable group of pKa approximately 9.5, previously identified in pH-jump studies of electron transfer, whose deprotonation must occur for the spin-interacting state to be established.     

FdC1, a novel ferredoxin protein capable of alternative electron partitioning, increases in conditions of acceptor limitation at photosystem I

In higher plants, [2Fe-2S] ferredoxin (Fd) proteins are the unique electron acceptors from photosystem I (PSI). Fds are soluble, and distribute electrons to many enzymes, including Fd:NADP(H) reductase (FNR), for the photoreduction of NADP(+). In addition to well studied [2Fe-2S] Fd proteins, higher plants also possess genes for significantly different, as yet uncharacterized Fd proteins, with extended C termini (FdCs). Whether these FdC proteins function as photosynthetic electron transfer proteins is not known. We examined whether these proteins play a role as alternative electron acceptors at PSI, using quantitative RT-PCR to follow how their expression changes in response to acceptor limitation at PSI, in mutant Arabidopsis plants lacking 90-95% of photosynthetic [2Fe-2S] Fd. Expression of the gene encoding one FdC protein, FdC1, was identified as being strongly up-regulated. We confirmed that this protein was chloroplast localized and increased in abundance on PSI acceptor limitation. We purified the recombinant FdC1 protein, which exhibited a UV-visible spectrum consistent with a [2Fe-2S] cluster, confirmed by EPR analysis. Measurements of electron transfer show that FdC1 is capable of accepting electrons from PSI, but cannot support photoreduction of NADP(+). Whereas FdC1 was capable of electron transfer with FNR, redox potentiometry showed that it had a more positive redox potential than photosynthetic Fds by around 220 mV. These results indicate that FdC1 electron donation to FNR is prevented because it is thermodynamically unfavorable. Based on our data, we speculate that FdC1 has a specific function in conditions of acceptor limitation at PSI, and channels electrons away from NADP(+) photoreduction.     

Interaction of iron-sulfur flavoprotein with oxygen and hydrogen peroxide

The dimeric iron-sulfur flavoprotein (Isf) from Methanosarcina thermophila contains one 4Fe-4S center and one FMN per monomer, and is the prototype of a family widely distributed among strictly anaerobic prokaryotes. Although Isf is able to oxidize ferredoxin, the physiological electron acceptor is unknown; thus, the ability of Isf to reduce O2 and H2O2 was investigated. The product of O2 or H2O2 reduction by Isf was determined to be water. The kinetic parameters of the oxidative half-reactions with O2 and H2O2 as electron acceptors were consistent with a role for Isf in combating oxidative stress. Isf depleted of the 4Fe-4S cluster was unable to oxidize ferredoxin and reduce the FMN cofactor, supporting a role for the cluster in transfer of electrons from ferredoxin to the cofactor. The implications of these properties on the possible function and mechanism of Isf are discussed.     

The flavoprotein subcomplex of complex I (NADH:ubiquinone oxidoreductase) from bovine heart mitochondria: insights into the mechanisms of NADH oxidation and NAD+ reduction from protein film voltammetry

Complex I (NADH:ubiquinone oxidoreductase) from bovine heart mitochondria contains 45 different subunits and nine redox cofactors. NADH is oxidized by a noncovalently bound flavin mononucleotide (FMN), then seven iron-sulfur clusters transfer the two electrons to quinone, and four protons are pumped across the inner mitochondrial membrane. Here, we use protein film voltammetry to investigate the mechanisms of NADH oxidation and NAD+ reduction in the simplest catalytically active subcomplex of complex I, the flavoprotein (Fp) subcomplex. The Fp subcomplex was prepared using chromatography and contained the 51 and 24 kDa subunits, the FMN, one [4Fe-4S] cluster, and one [2Fe-2S] cluster. The reduction potential of the FMN in the enzyme's active site is lower than that of free FMN (thus, the oxidized state of the FMN is most strongly bound) and close to the reduction potential of NAD+. Consequently, the catalytic transformation is reversible. Electrocatalytic NADH oxidation by subcomplex Fp can be explained by a model comprising substrate mass transport, the Michaelis-Menten equation, and interfacial electron transfer kinetics. The difference between the "catalytic" potential and the FMN potential suggests that the flavin is reoxidized before NAD+ is released or that intramolecular electron transfer from the flavin to the [4Fe-4S] cluster influences the catalytic rate. NAD+ reduction displays a marked activity maximum, below which the catalytic rate decreases sharply as the driving force increases. Two possible models reproduce the observed catalytic waveshapes: one describing an effect from reducing the proximal [2Fe-2S] cluster and the other the enhanced catalytic ability of the semiflavin state.     

Crystal Structure of Histamine Dehydrogenase from Nocardioides simplex

Histamine dehydrogenase (HADH) isolated from Nocardioides simplex catalyzes the oxidative deamination of histamine to imidazole acetaldehyde. HADH is highly specific for histamine, and we are interested in understanding the recognition mode of histamine in its active site. We describe the first crystal structure of a recombinant form of HADH (HADH) to 2.7-Å resolution. HADH is a homodimer, where each 76-kDa subunit contains an iron-sulfur cluster ([4Fe-4S]²⁺) and a 6-S-cysteinyl flavin mononucleotide (6-S-Cys-FMN) as redox cofactors. The overall structure of HADH is very similar to that of trimethylamine dehydrogenase (TMADH) from Methylotrophus methylophilus (bacterium W3A1). However, some distinct differences between the structure of HADH and TMADH have been found. Tyr⁶⁰, Trp²⁶⁴, and Trp³⁵⁵ provide the framework for the “aromatic bowl” that serves as a trimethylamine-binding site in TMADH is comprised of Gln⁶⁵, Trp²⁶⁷, and Asp³⁵⁸, respectively, in HADH. The surface Tyr⁴⁴² that is essential in transferring electrons to electron-transfer flavoprotein (ETF) in TMADH is not conserved in HADH. We use this structure to propose the binding mode for histamine in the active site of HADH through molecular modeling and to compare the interactions to those observed for other histamine-binding proteins whose structures are known.     

Deuterium isotope effects during carbon-hydrogen bond cleavage by trimethylamine dehydrogenase. Implications for mechanism and vibrationally assisted hydrogen tunneling in wild-type and mutant enzymes

His-172 and Tyr-169 are components of a triad in the active site of trimethylamine dehydrogenase (TMADH) comprising Asp-267, His-172, and Tyr-169. Stopped-flow kinetic studies with trimethylamine as substrate have indicated that mutation of His-172 to Gln reduces the limiting rate constant for flavin reduction approximately 10-fold (Basran, J., Sutcliffe, M. J., Hille, R., and Scrutton, N. S. (1999) Biochem. J. 341, 307-314). A kinetic isotope effect (KIE = k(H)/k(D)) accompanies flavin reduction by H172Q TMADH, the magnitude of which varies significantly with solution pH. With trimethylamine, flavin reduction by H172Q TMADH is controlled by a single macroscopic ionization (pK(a) = 6.8 +/- 0.1). This ionization is perturbed (pK(a) = 7.4 +/- 0.1) in reactions with perdeuterated trimethylamine and is responsible for the apparent variation in the KIE with solution pH. At pH 9.5, where the functional group controlling flavin reduction is fully ionized, the KIE is independent of temperature in the range 277-297 K, consistent with vibrationally assisted hydrogen tunneling during breakage of the substrate C-H bond. Y169F TMADH is approximately 4-fold more compromised than H172Q TMADH for hydrogen transfer, which occurs non-classically. Studies with Y169F TMADH suggest partial thermal excitation of substrate prior to hydrogen tunneling by a vibrationally assisted mechanism. Our studies illustrate the varied effects of compromising mutations on tunneling regimes in enzyme molecules.     

The electron transfer flavoprotein: ubiquinone oxidoreductases

Electron transfer flavoprotein: ubiqionone oxidoreductase (ETF-QO) is a component of the mitochondrial respiratory chain that together with electron transfer flavoprotein (ETF) forms a short pathway that transfers electrons from 11 different mitochondrial flavoprotein dehydrogenases to the ubiquinone pool. The X-ray structure of the pig liver enzyme has been solved in the presence and absence of a bound ubiquinone. This structure reveals ETF-QO to be a monotopic membrane protein with the cofactors, FAD and a [4Fe-4S](+1+2) cluster, organised to suggests that it is the flavin that serves as the immediate reductant of ubiquinone. ETF-QO is very highly conserved in evolution and the recombinant enzyme from the bacterium Rhodobacter sphaeroides has allowed the mutational analysis of a number of residues that the structure suggested are involved in modulating the reduction potential of the cofactors. These experiments, together with the spectroscopic measurement of the distances between the cofactors in solution have confirmed the intramolecular pathway of electron transfer from ETF to ubiquinone. This approach can be extended as the R. sphaeroides ETF-QO provides a template for investigating the mechanistic consequences of single amino acid substitutions of conserved residues that are associated with a mild and late onset variant of the metabolic disease multiple acyl-CoA dehydrogenase deficiency (MADD).     

Thermodynamic Redox Properties Governing the Half-Reduction Characteristics of Histamine Dehydrogenase from Nocardioides simplex

Histamine dehydrogenase from Nocardioides simplex is a homodimer and belongs to the family of iron-sulfur flavoproteins having one [4Fe-4S] cluster and one 6-S-cysteinyl FMN per monomer. In the reductive titration with histamine, two-electron reduction occurred per monomer at pH<9, while single-electron reduction proceeded at pH>9. The substrate-reduced histamine dehydrogenase yielded an electron paramagnetic resonance spectral signal assigned to the flavin semiquinone. The signal intensity increased with pH up to pH 9 and reached a maximum at pH>9. These unique features are explained in terms of the redox potential of the cofactors, where the redox potential was evaluated over a pH range from 7 to 10 by using a spectroelectrochemical titration method for the flavin and cyclic voltammetry for the [4Fe-4S] cluster. The bell-type pH dependence of the enzymatic activity is also discussed in terms of the pH dependence of the centers’ redox potential.     

Laser flash photolysis study of intermolecular and intramolecular electron transfer in trimethylamine dehydrogenase

Laser flash photolysis has been used to investigate the kinetics of reduction of trimethylamine dehydrogenase by substoichiometric amounts of 5-deazariboflavin semiquinone, and the subsequent intramolecular electron transfer from the FMN cofactor to the Fe4S4 center. The initial reduction event followed second-order kinetics (k = 1.0 x 10(8) M-1 s-1 at pH 7.0 and 6.4 x 10(7) M-1 s-1 at pH 8.5) and resulted in the formation of the neutral FMN semiquinone and the reduced iron-sulfur cluster (in a ratio of approximately 1:3). Following this, a slower, protein concentration independent (and thus intramolecular) electron transfer was observed corresponding to FMN semiquinone oxidation and iron-sulfur cluster reduction (k = 62 s-1 at pH 7.0 and 30 s-1 at pH 8.5). The addition of the inhibitor tetramethylammonium chloride to the reaction mixture had no effect on these kinetic properties, suggesting that this compound exerts its effect on the reduced form of the enzyme. Treatment of the enzyme with phenylhydrazine, which introduces a phenyl group at the 4a-position of the FMN cofactor, decreased both the rate constant for reduction of the protein and the extent of FMN semiquinone production, while increasing the amount of iron-sulfur center reduction, consistent with the results obtained with the native enzyme. Experiments in which the kinetics of reduction of the enzyme were determined during various stages of partial reduction were also consistent with these results, and further indicated that the FMN semiquinone form of the enzyme is more reactive toward the deazariboflavin reductant than is the oxidized FMN.(ABSTRACT TRUNCATED AT 250 WORDS)