Reactions of electron-transfer flavoprotein and electron-transfer flavoprotein: ubiquinone oxidoreductase.

Electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF-Q oxidoreductase) catalyses the re-oxidation of reduced electron-transfer flavoprotein (ETF) with ubiquinone-1 (Q-1) as the electron acceptor. A kinetic assay for the enzyme was devised in which glutaryl-CoA in the presence of glutaryl-CoA dehydrogenase was used to reduce ETFox. and the reduction of Q-1 was monitored at 275 nm. The partial reactions involved in the overall assay system were examined. Glutaryl-CoA dehydrogenase catalyses the rapid reduction of ETFox. to the anionic semiquinone (ETF.-), but reduces ETF.- to the fully reduced form (ETFhq) at a rate that is about 6-fold lower. ETF.-, but not ETFhq, is directly re-oxidized by Q-1 at a rate that, depending on the steady-state concentration of ETF.-, may contribute significantly to the overall reaction. ETF-Q oxidoreductase catalyses rapid disproportionation of ETF.- with an equilibrium constant of about 1.0 at pH 7.8. In the presence of Q-1 it also catalyses the re-oxidation of ETFhq at a rate that is faster than that of the overall reaction. Rapid-scan experiments indicated the formation of ETF.-, but its fractional concentration in the early stages of the re-oxidation of ETFhq is low. The data indicate that the re-oxidation of ETFhq proceeds at a rate that is adequate to account for the overall rate of electron transfer from glutaryl-CoA to Q-1. An unusual property of ETF-Q oxidoreductase seems to be that it not only catalyses the re-oxidation of the reduced forms of ETF but also facilitates the complete reduction of ETFox. to ETFhq by disproportionation of the radical.     

Tryptophan fluorescence in electron-transfer flavoprotein:ubiquinone oxidoreductase: fluorescence quenching by a brominated pseudosubstrate

We have studied the intrinsic fluorescence of the 12 tryptophan residues of electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO). The fluorescence emission spectrum (lambda ex 295 nm) showed that the fluorescence is due to the tryptophan residues and that the contribution of the 22 tyrosine residues is minor. The emission maximum (lambda m 334 nm) and the bandwidth (delta lambda 1/2 56 nm) suggest that the tryptophans lie in hydrophobic environments in the oxidized protein. Further, these tryptophans are inaccessible to a range of ionic and nonionic collisional quenching agents, indicating that they are buried in the protein. Enzymatic or chemical reduction of ETF:QO results in a 5% increase in fluorescence with no change of lambda m or delta lambda 1/2. This change is reversible upon reoxidation and is likely to reflect a conformational change in the protein. The ubiquinone analogue Q0(CH2)10Br, a pseudosubstrate of ETF:QO (Km = 2.6 microM; kcat = 210 s-1), specifically quenches the fluorescence of one tryptophan residue (Kd = 1.6-3.2 microM) in equilibrium fluorescence titrations. The ubiquinone homologue UQ-2 (Km = 2 microM; kcat = 162 s-1) and the analogue Q0(CH2)10OH (Km = 2 microM; kcat = 132 s-1) do not quench tryptophan fluorescence; thus the brominated analogue acts as a static heavy atom quencher. We also describe a rapid purification for ETF:QO based on extraction of liver submitochondrial particles with Triton X-100 and three chromatographic steps, which results in yields 3 times higher than previously published methods.     

Alternate electron acceptors for medium-chain acyl-CoA dehydrogenase: use of ferricenium salts

Medium-chain acyl-CoA dehydrogenase reduced with octanoyl-CoA is reoxidized in two one-electron steps by two molecules of the physiological oxidant, electron transferring flavoprotein (ETF). The organometallic oxidant ferricenium hexafluorophosphate (Fc+PF6-) is an excellent alternative oxidant of the dehydrogenase and mimics a number of the features shown by ETF. Reoxidation of octanoyl-CoA-reduced enzyme (200 microM Fc+PF6- in 100 mM Hepes buffer, pH 7.6, 1 degree C) occurs in two one-electron steps with pseudo-first-order rate constants of 40 s-1 and about 200 s-1 for k1 and k2, respectively. The reaction is comparatively insensitive to ionic strength, and evidence of rate saturation is encountered at high ferricenium ion concentration. As observed with ETF, the free two-electron-reduced dehydrogenase is a much poorer kinetic reductant of Fc+PF6-, with rate constants of 3 s-1 and 0.3 s-1 (for k1 and k2, respectively) using 200 microM Fc+PF6-. In addition to the enoyl-CoA product formed during the dehydrogenation of octanoyl-CoA, binding a number of redox-inert acyl-CoA analogues (notably 3-thia- and 3-oxaoctanoyl-CoA) significantly accelerates electron transfer from the dehydrogenase to Fc+PF6-. Those ligands most effective at accelerating electron transfer favor deprotonation of reduced flavin species in the acyl-CoA dehydrogenase. Thus this rate enhancement may reflect the anticipated kinetic superiority of anionic flavin forms as reductants in outer-sphere electron-transfer processes. Evidence consistent with the presence of two distinct loci for redox communication with the bound flavin in the acyl-CoA dehydrogenase is presented.     

Preferential cross-linking of the small subunit of the electron-transfer flavoprotein to general acyl-CoA dehydrogenase.

The interaction between pig liver mitochondrial electron-transfer flavoprotein (ETF) and general acyl-CoA dehydrogenase (GAD) was investigated by means of the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)propionate. Neither ETF or GAD contained reactive thiol groups. The substitution of 9.4 lysine residues/FAD group in GAD with pyridyl disulphide structures did not affect the catalytic activity of the enzyme. Thiol groups were introduced into ETF by thiolation with methyl 4-mercaptobutyrimidate. ETF containing 10.5 reactive thiol groups/FAD group showed undiminished electron-acceptor activity with respect to GAD. The reaction of thiolated ETF and GAD containing pyridyl disulphide structures resulted in a decreased staining intensity of the small subunit of ETF on SDS/polyacrylamide-gel electrophoresis. Preferential cross-linking of the smaller subunit of ETF to GAD did not take place when ETF was first treated with SDS, but was unaffected by reduction of GAD by octanoyl-CoA.     

Energetics of beta-oxidation. Reduction potentials of general fatty acyl-CoA dehydrogenase, electron transfer flavoprotein, and fatty acyl-CoA substrates

We have determined reduction potentials for porcine mitochondrial general fatty acyl-CoA dehydrogenase (GAD) and electron transfer flavoprotein (ETF) using an anaerobic spectroelectrochemical titration method. Computer simulation techniques were used to analyze the absorbance data. Nernst plots of the simulated data gave E'0, 7.1, quinone/semiquinone = -0.014 V and E'0, 7.1, semiquinone/hydroquinone = -0.036 V for ETF and E'0, 7.1, quinone/semiquinone = -0.155 V and E'0, 7.1, semiquinone/hydroquinone = -0.122 V for GAD. Using these techniques we have also determined a conditional reduction potential of -0.156 V for the chromophore producing fatty acyl-CoA substrate beta-2-furylpropionyl-CoA. From this value and our previous determination of the equilibrium constant for the transhydrogenation reaction between beta-2-furylpropionyl-CoA and the oxidized substrate crotonyl-CoA (Keq = 10.4), we have determined a reduction potential of -0.126 V for the butyryl-CoA/crotonyl-CoA couple. In light of the structural similarity between butyryl-CoA and octanoyl-CoA, the optimal substrate for GAD, the reduction potential for octanoyl-CoA should be similar to that for butyryl-CoA; i.e. fatty acyl-CoA substrates and GAD are essentially isopotential. The ability of octanoyl-CoA to reduce GAD quantitatively (Keq = 9.0) poses a dilemma in light of the nearly equal reduction potentials. We postulate that the stable charge-transfer complex formed between enzyme and optimal product is significantly lower in energy than enzyme and product and thus is responsible for pulling the reaction toward completion.     

Electron-transfer flavoprotein-ubiquinone oxidoreductase from pig liver: purification and molecular, redox, and catalytic properties

Electron-transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) was purified to homogeneity from pig liver submitochondrial particles. It is comparable in molecular weight and general properties to ETF-QO from beef heart [Ruzicka, F. J., & Beinert, H. (1977) J. Biol. Chem. 252, 8440-8445], and the electron spin resonance signals of the reduced iron-sulfur cluster are essentially identical. ETF-QO catalyzes the transfer of electrons from electron-transfer flavoprotein (ETF) to nitro blue tetrazolium, with a sluggish reaction turnover number of about 10-30 min-1. In contrast, the enzyme rapidly disproportionates ETF semiquinone, with a turnover number of 200 s-1. The reverse reaction, comproportionation of oxidized and hydroquinone ETF, provides an enzymatic assay for ETF-QO with picomolar sensitivity. Equilibrium spectrophotometric titrations show that ETF-QO accepts a maximum of two electrons from ETF and accepts three electron equivalents from dithionite or by photochemical reduction. All electrons from the enzymatically or chemically reduced protein can be transferred to 2,3-dimethoxy-5-methyl-6-pentyl-1,4-benzoquinone (PB), and this reaction is readily reversible. Reduction of ETF-QO by 2,3-dimethoxy-5-methyl-6-pentyl-1,4-benzohydroquinone is pH dependent and indicates the enzyme to have a redox potential that decreases by 47 mV per pH unit. Therefore, ETF-QO binds one to two protons upon reduction. The EO' at pH 7.3 is 38 mV. The ability of ETF-QO to catalyze the equilibration of ETF redox states has been used to evaluate the equilibrium 2ETFsq + nH+ in equilibrium ETFox + ETFhq.(ABSTRACT TRUNCATED AT 250 WORDS)     

Unusual redox properties of electron-transfer flavoprotein from Methylophilus methylotrophus

The most positive redox potential ever recorded for a flavin adenine dinucleotide (FAD) containing protein has been measured for an electron-transfer flavoprotein (ETF) synthesized by Methylophilus methylotrophus. This potential value, 0.196 V versus the standard hydrogen electrode (vs SHE), was measured at pH 7.0 for the one-electron reduction of fully oxidized ETF (ETFox) to the red anionic semiquinone form of ETF (ETF.-). Quantitative formation of ETF.- was observed. The first successful reduction of ETF from M. methylotrophus to its two-electron fully reduced form was also achieved. Although addition of the second electron to ETF.- was extremely slow, the potential value measured for this reduction was -0.197 V vs SHE, suggesting a kinetic rather than thermodynamic barrier to two-electron reduction. These data are believed to be consistent with the postulated catalytic function of ETF to accept one electron from the iron-sulfur cluster of trimethylamine dehydrogenase (TMADH). The second electron reduction appears to have no catalytic function. The very positive potential measured for this ETF and the wide separation of potentials for the two electron reduction steps show that this ETF is a unique and interesting flavoprotein. In addition, this work highlights that while ETFs exhibit similar structural and spectral properties, they display wide variations in redox properties.     

Conformational change accompanies redox reactions of the tetraheme cytochrome c-554 of Nitrosomonas europaea

Cytochrome c-554 of the ammonia-oxidizing chemolithoautotropic bacteria is thought to mediate electron transfer from hydroxylamine oxidoreductase to a terminal oxidase and/or to ammonia monooxygenase. The cytochrome has four c hemes which interact magnetically and have the same redox potential. We report that the kinetics of reduction of ferric cytochrome c-554 by dithionite or the oxidation of ferrous cytochrome c-554 by O2 or H2O2 are complex and multiphasic. Transient rapid-scan difference spectra indicate discrete maxima at approximately 418 nm, 425 nm and 432 nm. Absorbance changes at all three difference maxima appear to occur in all kinetic phases, although not in equal amounts for each wavelength. Reduction by 20 mM dithionite was biphasic. At pH 7.5 the first phase, which involved approximately 50% of the total absorbance change, had a rate constant (20 degrees C) of 140 s-1 and energy of activation of 20 kJ X mol-1. The slow phase had a rate constant 0.43 s-1 and a relatively high energy of activation, 87 kJ X mol-1, suggesting that a change in protein configuration accompanied the reaction. As the pH of the solution increased, the rate constant for both phases decreased and the fraction of absorbance change in the rapid phase increased. Oxidation of ferrous cytochrome c-554 by O2 involved a discrete rapid phase with a rate constant of 14 s-1, accounting for 6% of the absorbance. The remainder of the reaction was multiphasic with rate constants in the range 0.1-0.01 s-1. With H2O2 as the oxidant, the rapid phase involved 39% of the change in absorbance with a rate constant of 19 s-1. The remainder of the reoxidation was multiphasic with rate constants ranging over 0.4-0.01 s-1.     

Redox properties of electron-transferring flavoprotein from Megasphaera elsdenii

Electron-transferring flavoprotein (ETF) from the anaerobic bacterium Megasphaera elsdenii catalyzes electron transfer from NADH or D-lactate dehydrogenase to butyryl-CoA dehydrogenase. As a basis for understanding the interactions of ETF with its substrates, we report here on the redox properties of ETF alone. ETF exhibited reversible, two-electron transfer during electrochemical reduction in the presence of mediator dyes. The midpoint redox potentials of the FAD cofactor were -0.185 V at pH 5.5, -0.259 V at pH 7.1 and -0.269 +/- 0.013 V at pH 8.4, all versus the standard hydrogen electrode In the presence of the indicator dye 1-deazariboflavin, the Nernst slopes were 0.029 V and 0.026 V at pH 5.5 and pH 7.1, respectively, compared with an expected value of 0.028 V at 10 degrees C. At pH 8.4, in the presence of 2-hydroxy-1,4-naphthoquinone or phenosafranine, the Nernst slope varied from 0.021 V to 0.041 V. In the experiments at pH 8.4, equilibration was very slow in the reductive direction and a difference of as much as 30 mV was observed between reductive and oxidative midpoints. ETF exhibited no thermodynamic stabilization of the radical form of the FAD cofactor during electrochemical reduction at pH 5.5, 7.1 or 8.4. However, up to 93% of kinetically stable, anionic radical was produced by dithionite titration at pH 8.5. Molar absorptivities of ETF radical were 17,000 M-1 X cm-1 at 365 nm and 5100 M-1 X cm-1 at 450 nm. The four ETF preparations used here contained less than 7% 6-OH-FAD. However, two of the preparations contained significant amounts (up to 30%) of flavin which stabilized radical and reduced at potentials 0.2 V more positive than those required for reduction of the major form of ETF. This is referred to as the B form of ETF. The proportion of ETF-FAD in the B form was increased by incubation with free FAD or by a cycle of reduction and reoxidation. These treatments caused marked changes in the absorption spectrum of oxidized ETF and decreases of 20-25% in ETF units/A450.     

Cross-linking of the electron-transfer flavoprotein to electron-transfer flavoprotein-ubiquinone oxidoreductase with heterobifunctional reagents.

The mitochondrial electron-transfer flavoprotein (ETF) is a heterodimer containing only one FAD. In previous work on the structure-function relationships of ETF, its interaction with the general acyl-CoA dehydrogenase (GAD) was studied by chemical cross-linking with heterobifunctional reagents [D. J. Steenkamp (1987) Biochem. J. 243, 519-524]. GAD whose lysine residues were substituted with 3-(2-pyridyldithio)propionyl groups was preferentially cross-linked to the small subunit of ETF, the lysine residues of which had been substituted with 4-mercaptobutyramidine (MBA) groups. This work was extended to the interaction of ETF with ETF-ubiquinone oxidoreductase (ETF-Q ox). ETF-Q ox was partially inactivated by modification with N-succinimidyl 3-(2-pyridyldithio)propionate to introduce pyridyl disulphide structures. A similar modification of ETF caused a large increase in the apparent Michaelis constant of ETF-Q ox for modified ETF owing to the loss of positive charge on some critical lysines of ETF. When ETF-Q ox was modified with 2-iminothiolane to introduce 4-mercaptobutyramidine groups, only a minor effect on the activity of the enzyme was observed. To retain the positive charges on the lysine residues of ETF, pyridyl disulphide structures were introduced by treating ETF with 2-iminothiolane in the presence of 2,2'-dithiodipyridyl. The electron-transfer activity of the resultant ETF preparation containing 4-(2-pyridyldithio)butyramidine (PDBA) groups was only slightly affected. When ETF-Q ox substituted with MBA groups was mixed with ETF bearing PDBA groups, at least 70% of the cross-links formed between the two proteins were between the small subunit of ETF and ETF-Q ox. ETF-Q ox, therefore, interacts predominantly with the same subunit of ETF as GAD. Variables which affect the selectivity of ETF-Q ox cross-linking to the subunits of ETF are considered.     

Kinetic mechanism of Tritrichomonas foetus inosine 5'-monophosphate dehydrogenase

IMP dehydrogenase (IMPDH) catalyzes the oxidation of IMP to XMP with conversion of NAD+ to NADH. This reaction is the rate-limiting step in de novo guanine nucleotide biosynthesis. IMPDH is a target for antitumor, antiviral, and immunosuppressive chemotherapy. We have determined the complete kinetic mechanism for IMPDH from Tritrichomonas foetus using ligand binding, isotope effect, pre-steady-state kinetic, and rapid quench kinetic experiments. Both substrates bind to the free enzyme, which suggests a random mechanism. IMP binds to the enzyme in two steps. Two steps are also involved when IMP binds to a mutant IMPDH in which the active site Cys is substituted with a Ser. This observation suggests that this second step may be a conformational change of the enzyme. No Vm isotope effect is observed when [2-2H]IMP is the substrate which indicates that hydride transfer is not rate-limiting. This result is confirmed by the observation of a pre-steady-state burst of NADH production when monitored by absorbance. However, when NADH production was monitored by fluorescence, the rate constant for the exponential phase is 5-10-fold lower than when measured by absorbance. This observation suggests that the fluorescence of enzyme-bound NADH is quenched and that this transient represents NADH release from the enzyme. The time-dependent formation and decay of [14C]E-XMP intermediates was monitored using rapid quench kinetics. These experiments indicate that both NADH release and E-XMP hydrolysis are rate-limiting and suggest that NADH release precedes hydrolysis of E-XMP.     

Reaction of electron-transfer flavoprotein with electron-transfer flavoprotein-ubiquinone oxidoreductase

The oxidative half-reaction of electron-transfer flavoprotein (ETF), electron transfer from ETF to electron-transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO), is dependent on complementary surface charges on the two proteins. ETF is the positively charged member of the redox pair. The evidence is based on the pH and ionic strength dependencies of the comproportionation of oxidized ETF and ETF hydroquinone catalyzed by ETF-QO and on the effects of chemical modification of ETF on the comproportionation reaction. Acetylation of one and five epsilon-amino groups of lysyl residues results in 3- and 13-fold increases, respectively, in the Km of ETF-QO for ETF but no change in Vmax. Amidination, which maintains positive charge at modified loci, has no effect on steady-state kinetic constants. These chemical modifications have no effect on the equilibrium constant for equilibration of ETF redox states. The Km of ETF-QO for ETF is pH dependent above pH 8.5, suggesting titration of lysyl residues as previously observed in studies of the reductive half-reaction of ETF [Beckmann, J. D., & Frerman, F. E. (1983) J. Biol. Chem. 258, 7563-7569]. The ionic strength dependence of TN/KmETF for the reaction follows the limiting Br?nsted equation ln (TN/Km) = ln k0 + 2 alpha Z1Z2I1/2, and Z1Z2, the product of charges on the reacting proteins, is similar to the value of Z1Z2 for the reductive half-reaction of ETF by the general acyl-CoA dehydrogenase. The ETF-QO-catalyzed comproportionation reaction exhibits a primary deuterium isotope effect in D2O, perhaps indicating the participation of solvent water in the electron-transfer reaction.     

Kinetic studies of Rhus vernicifera laccase. Evidence for multi-electron transfer and an oxygen intermediate in the reoxidation reaction.

1. The reoxidation of reduced Rhus vernicifera laccase (monophenol,dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) by molecular oxygen has been studied by optical absorption and EPR methods. 2. The reoxidation by oxygen of the type 1 Cu+ and the two-electron acceptor is characterized by a second-order rate constant of about 5-10(6) M-1-s-1. 3. The appearance of an optical intermediate (with an absorbance maximum around 360 nm) parallels the reoxidation of type 1 Cu+ and the two-electron acceptor. It disappears in a first-order reaction with a half-time of 20 s. A similar intermediate is formed during normal turnover. 4. The type 2 Cu+ appears to be reoxidized in an intramolecular reaction with a half-time of about 20 s, suggesting a correlation between the reoxidation of this site and the disappearance of the optical intermediate. 5. The results suggest that three electrons are rapidly transferred to oxygen leading to the formation of an enzyme-bound oxygen intermediate.     

Crystal structure of Paracoccus denitrificans electron transfer flavoprotein: structural and electrostatic analysis of a conserved flavin binding domain

The crystal structure of electron transfer flavoprotein (ETF) from Paracoccus denitrificans was determined and refined to an R-factor of 19.3% at 2.6 A resolution. The overall fold is identical to that of the human enzyme, with the exception of a single loop region. Like the human structure, the structure of the P. denitrificans ETF is comprised of three distinct domains, two contributed by the alpha-subunit and the third from the beta-subunit. Close analysis of the structure reveals that the loop containing betaI63 is in part responsible for conferring the high specificity of AMP binding by the ETF protein. Using the sequence and structures of the human and P. denitrificans enzymes as models, a detailed sequence alignment has been constructed for several members of the ETF family, including sequences derived for the putative FixA and FixB proteins. From this alignment, it is evident that in all members of the ETF family the residues located in the immediate vicinity of the FAD cofactor are identical, with the exception of the substitution of serine and leucine residues in the W3A1 ETF protein for the human residues alphaT266 and betaY16, respectively. Mapping of ionic differences between the human and P. denitrificans ETF onto the structure identifies a surface that is electrostatically very similar between the two proteins, thus supporting a previous docking model between human ETF and pig medium-chain acyl-CoA dehydrogenase (MCAD). Analysis of the ionic strength dependence of the electron transfer reaction between either human or P. denitrificans ETF and MCAD demonstrates that the human ETF functions optimally at low ( approximately 10 mequiv) ionic strength, while P. denitrificans ETF is a better electron acceptor at higher (>75 mequiv) ionic strength. This suggests that the electrostatic surface potential of the two proteins is very different and is consistent with the difference in isoelectric points between the proteins. Analysis of the electrostatic potentials of the human and P. denitrificans ETFs reveals that the P. denitrificans ETF is more negatively charged. This excess negative charge may contribute to the difference in redox potentials between the two ETF flavoproteins and suggests an explanation for the opposing ionic strength dependencies for the reaction of MCAD with the two ETFs. Furthermore, by analysis of a model of the previously described human-P. denitrificans chimeric ETF protein, it is possible to identify one region of ETF that participates in docking with ETF-ubiquinone oxidoreductase, the physiological electron acceptor for ETF.     

Partial purification and characterization of glutaryl-coenzyme A dehydrogenase, electron transfer flavoprotein, and electron transfer flavoprotein-Q oxidoreductase from Paracoccus denitrificans.

Glutaryl-coenzyme A (CoA) dehydrogenase and the electron transfer flavoprotein (ETF) of Paracoccus denitrificans were purified to homogeneity from cells grown with glutaric acid as the carbon source. Glutaryl-CoA dehydrogenase had a molecular weight of 180,000 and was made up of four identical subunits with molecular weights of about 43,000 each of which contained one flavin adenine dinucleotide molecule. The enzyme catalyzed an oxidative decarboxylation of glutaryl-CoA to crotonyl-CoA, was maximally stable at pH 5.0, and lost activity readily at pH values above 7.0. The enzyme had a pH optimum in the range of 8.0 to 8.5, a catalytic center activity of about 960 min-1, and apparent Michaelis constants for glutaryl-CoA and pig liver ETF of about 1.2 and 2.5 microM, respectively. P. denitrificans ETF had a visible spectrum identical to that of pig liver ETF and was made up of two subunits, only one of which contained a flavin adenine dinucleotide molecule. The isoelectric point of P. denitrificans ETF was 4.45 compared with 6.8 for pig liver ETF. P. denitrificans ETF accepted electrons not only from P. denitrificans glutaryl-CoA dehydrogenase, but also from the pig liver butyryl-CoA and octanoyl-CoA dehydrogenases. The apparent Vmax was of similar magnitude with either pig liver or P. denitrificans ETF as an electron acceptor for these dehydrogenases. P. denitrificans glutaryl-CoA dehydrogenase and ETF were used to assay for the reduction of ubiquinone 1 by ETF-Q oxidoreductase in cholate extracts of P. denitrificans membranes. The ETF-Q oxidoreductase from P. denitrificans could accept electrons from either the bacterial or the pig liver ETF. In either case, the apparent Km for ETF was infinitely high. P. denitrificans ETF-Q oxidoreductase was purified from contaminating paramagnets, and the resultant preparation had electron paramagnetic resonance signals at 2.081, 1.938, and 1.879 G, similar to those of the mitochondrial enzyme.     

Structural intermediates in folding of yeast iso-2 cytochrome c

The kinetic properties of the folding reactions of iso-2 cytochrome c from Saccharomyces cerevisiae have been investigated by stopped-flow and temperature-jump methods. Three different structural probes are compared: (1) absorbance changes in the visible reflecting changes in heme environment, (2) ultraviolet absorbance changes due to the exposure of aromatic groups to solvent, and (3) tryptophan fluorescence attributable principally to the average distance between the tryptophan residue (donor) and the heme (quencher). In addition, two probes either indicative of or correlated with function, ascorbic acid reducibility and the 695-nm absorbance band, have been used to monitor specifically the rate of formation of the native protein on refolding. The fastest phase observed (tau 3) has a measurable relative amplitude only when monitored by visible absorbance changes, suggesting that this reaction involves changes in heme environment in the absence of significant changes in the heme to tryptophan distance or in the extent to which aromatic groups are exposed to solvent. Different slow phases are observed when complete refolding is monitored by visible or ultraviolet absorbance (tau 1a) as opposed to tryptophan fluorescence (tau 1b), the fluorescence changes being complete on a time scale 4-8-fold faster than for absorbance. A mid-range kinetic phase (tau 2) is detected by all three structural probes. When ascorbic acid reducibility or 695-nm absorbance changes are used to monitor the rate of formation of the native protein, two phases are detected: tau 2 and tau 1a. Taken together these results demonstrate that kinetic phase tau 1b results in the formation of a structural intermediate in folding with fluorescence close to that of the native protein but with distinct absorbance properties.     

Kinetic mechanism of the interaction of D-cycloserine with serine hydroxymethyltransferase

The kinetic mechanism for the interaction of D-cycloserine with serine hydroxymethyltransferase (EC 2.1.2.1) from sheep liver was established by measuring changes in the activity, absorbance, and circular dichoism (CD) of the enzyme. The irreversible inhibition of the enzyme was characterized by three detectable steps: an initial rapid step followed by two successive steps with rate constants of 5.4 X 10(-3) s-1 and 1.4 X 10(-4) s-1. The first step was distinguished by a rapid disappearance of the enzyme absorbance peak at 425 nm, a decrease in the enzyme activity to 25% of the uninhibited velocity, and a lowering of the CD intensity at 432 nm to about 65% of the original value. The second step of the interaction was accompanied by a complete loss of enzyme activity and a marginal increase in the CD intensity at 432 nm. The final step resulted in the complete loss of the enzyme absorbance at 425 nm and of the CD band at 432 nm. The products of the reaction were identified as (a) apoenzyme by absorbance measurements, CD spectra, and reconstitution with pyridoxal 5'-phosphate and (b) a pyridoxal 5'-phosphate-D-cycloserine Schiff's base complex identified by its fluorescence and absorbance spectra. The Schiff base complex was expelled from the enzyme active site in the final step of the reaction. The proposed mechanism, which is different from those operative in other pyridoxal phosphate dependent enzymes, probably accounts for the selective inhibition of serine hydroxymethyltransferase by the drug in vivo.     

Delayed fluorescence from Rhodopseudomonas sphaeroides following single flashes.

Delayed fluorescence from Rhodopseudomonas sphaeroides chromatophores was studied with the use of short flashes for excitation. Although the delayed fluorescence probably arises from a back-reaction between the oxidized reaction center bacteriochlorophyll complex (P+) and the reduced electron acceptor (X-), the decay of delayed fluorescence after a flash is much faster (tau1/2 approximately 120 mus) than the decay of P+X-. The rapid decay of delayed fluorescence is not due to the uptake of a proton from the solution, nor to a change in membrane potential. It correlates with small optical absorbance changes at 450 and 770 nm which could reflect a change in the state of X-. The intensity of the delayed fluorescence is 11-18-fold greater if the excitation flashes are spaced 2 s apart than it is if they are 30 s apart. The enhancement of delayed fluorescence at high flash repetition rates occurs only at redox potentials which are low enough (less than +240 mV) so that electron donors are available to reduce P+X- to PX- in part of the reaction center population. The enhancement decays between flashes as PX- is reoxidized to PX, as measured by the recovery of photochemical activity. Evidently, the reduction of P+X- to PX- leads to the storage of free energy that can be used on a subsequent flash to promote delayed fluorescence. The reduction of P+X- also is associated with a carotenoid spectral shift which decays as PX- is reoxidized to PX. Although this suggests that the free energy which supports the delayed fluorescence might be stored as a membrane potential, the ionophore gramicidin D only partially inhibits the enhancement of delayed fluorescence. With widely separated flashes, gramicidin has no effect on delayed fluorescence. At redox potentials low enough to keep X fully reduced, delayed fluorescence of the type described above does not occur, but one can detect weak luminescence which probably is due to phosphorescence of a protoporphyrin.     

The lipoamide dehydrogenase from Mycobacterium tuberculosis permits the direct observation of flavin intermediates in catalysis

Lipoamide dehydrogenase catalyses the NAD(+)-dependent oxidation of the dihydrolipoyl cofactors that are covalently attached to the acyltransferase components of the pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, and glycine reductase multienzyme complexes. It contains a tightly, but noncovalently, bound FAD and a redox-active disulfide, which cycle between the oxidized and reduced forms during catalysis. The mechanism of reduction of the Mycobacterium tuberculosis lipoamide dehydrogenase by NADH and [4S-(2)H]-NADH was studied anaerobically at 4 degrees C and pH 7.5 by stopped-flow spectrophotometry. Three phases of enzyme reduction were observed. The first phase, characterized by a decrease in absorbance at 400-500 nm and an increase in absorbance at 550-700 nm, was fast (k(for) = 1260 s(-)(1), k(rev) = 590 s(-)(1)) and represents the formation of FADH(2).NAD(+), an intermediate that has never been observed before in any wild-type lipoamide dehydrogenase. A primary deuterium kinetic isotope effect [(D)(k(for) + k(rev)) approximately 4.2] was observed on this phase. The second phase, characterized by regain of the absorbance at 400-500 nm, loss of the 550-700 nm absorbance, and gain of 500-550 nm absorbance, was slower (k(obs) = 200 s(-)(1)). This phase represents the intramolecular transfer of electrons from FADH(2) to the redox-active disulfide to generate the anaerobically stable two-electron reduced enzyme, EH(2). The third phase, characterized by a decrease in absorbance at 400-550 nm, represents the formation of the four-electron reduced form of the enzyme, EH(4). The observed rate constant for this phase showed a decreasing NADH concentration dependence, and results from the slow (k(for) = 57 s(-)(1), k(rev) = 128 s(-)(1)) isomerization of EH(2) or slow release of NAD(+) before rapid NADH binding and reaction to form EH(4). The mechanism of oxidation of EH(2) by NAD(+) was also investigated under the same conditions. The 530 nm charge-transfer absorbance of EH(2) shifted to 600 nm upon NAD(+) binding in the dead time of mixing of the stopped-flow instrument and represents formation of the EH(2).NAD(+) complex. This was followed by two phases. The first phase (k(obs) = 750 s(-)(1)), characterized by a small decrease in absorbance at 435 and 458 nm, probably represents limited accumulation of FADH(2).NAD(+). The second phase was characterized by an increase in absorbance at 435 and 458 nm and a decrease in absorbance at 530 and 670 nm. The observed rate constant that describes this phase of approximately 115 s(-)(1) probably represents the overall rate of formation of E(ox) and NADH from EH(2) and NAD(+), and is largely determined by the slower rates of the coupled sequence of reactions preceding flavin oxidation.     

Changes in the fluorescence of bound nucleotide during the reaction catalysed by glucose-fructose oxidoreductase from Zymomonas mobilis.

The reduction of gluconolactone by glucose-fructose oxidoreductase containing tightly bound NADPH (enzyme-NADPH) is biphasic in nucleotide fluorescence. The initial rapid decrease, which represents quenching of the fluorescence by bound lactone, is followed by a slower decrease which corresponds to the change in absorbance. At low glucose concentrations, the oxidation of glucose by enzyme-NADP+ involves a single first-order process with similar rate constants in fluorescence and absorbance. At higher glucose concentrations, the apparent first-order rate constants for the fluorescence change are less than those for the absorbance change. This is consistent with a mechanism in which the fluorescence change occurs during the lactone dissociation step, which is slower than the hydrogen transfer step during which the absorbance change occurs. The rate constant for gluconolactone dissociation is 360 +/- 10 s-1 and this step is therefore rate-determining for the overall reaction. Reduction of fructose by enzyme-NADPH is first order with a limiting rate constant of at least 2000 s-1.