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The fluorescence decay curves of the flavin in all pyruvate dehydrogenase complexes studied here are consistent with a two-exponential fit. One of the lifetimes calculated is very short, as demonstrated by experiments in which a mode-locked argon-ion laser was used for excitation. In three complexes out of the four which were investigated, about equal weights for the amplitudes of the two lifetimes are found. In the three-component complex from Azotohacter vinelundii this is not the case. No effects of the protein concentration on the lifetimes of the fluorophore were found in the concentration range studied. A small but significant difference in lifetime is observed for the A . virzelundii complexes when coenzyme-free complex is compared with complex to which Mg2 + and thiamin diphosphate are added. The correlation time calculated from the polarized decay of the flavin fluorescence at 11 ' C is around 40 ns and 50 ns for A. vinelandii complexes and Escherichia coli complexes respectively. This correlation time is of the same order as the rotational correlation time of free lipoamide dehydrogenase itself, but much shorter than would be expected from the molecular weights of the complexes. Models explaining the two lifetimes are discussed. A catalytic mechanism based on the internal mobility of the lipoamide dehydrogenase inside the multi-enzyme complex is proposed.A number of fluorescence studies on flavinadenine dinucleotide either free in solution or bound to a protein have been reported [I -61. Changes in fluorescence properties were used to detect conformational changes. Energy transfer studies to and from the flavin in lipoamide dehydrogenase and in the pyruvate dehydrogenase complex have been applied to obtain distance information in proteins and protein complexes. Natural donors, like tryptophan [2] as well as covalently bound fluorescent labels [7-101, have been used for this purpose.Previous lifetime studies employing the singlephoton counting technique on lipoamide dehydrogenase from pig heart by Visser et al. [ I l l revealed that the fluorescence decay of the bound flavins was consistent with the presence of two lifetimes. This result was confirmed by phase fluorometer data showing non-identical apparent lifetimes from phase and modulation [12]. Since lipoamide dehydrogenase is an enzyme consisting of two identical peptide chains and since the amplitudes as determined by single-~~ Ahhreviutzon TPP, thiamin diphosphate.photon counting were about identical, it was proposed that the two monomers have bound their respective FAD in a slightly different manner.The presence of two lifetimes is not only interesting, but also complicates considerably the conclusions from, for example, energy transfer. For this reason phase and modulation lifetimes of FAD in lipoamide dehydrogenase free and bound to pyruvate dehydrogenase complexes from other sources were compared [9,12]. The FAD in pyruvate dehydrogenase complex isolated from pig heart appeared to show even bigger differences in phase and modulation lifetimes...
The fluorescence decay curves of the flavin in all pyruvate dehydrogenase complexes studied here are consistent with a two-exponential fit. One of the lifetimes calculated is very short, as demonstrated by experiments in which a mode-locked argon-ion laser was used for excitation. In three complexes out of the four which were investigated, about equal weights for the amplitudes of the two lifetimes are found. In the three-component complex from Azotohacter vinelundii this is not the case. No effects of the protein concentration on the lifetimes of the fluorophore were found in the concentration range studied. A small but significant difference in lifetime is observed for the A . virzelundii complexes when coenzyme-free complex is compared with complex to which Mg2 + and thiamin diphosphate are added. The correlation time calculated from the polarized decay of the flavin fluorescence at 11 ' C is around 40 ns and 50 ns for A. vinelandii complexes and Escherichia coli complexes respectively. This correlation time is of the same order as the rotational correlation time of free lipoamide dehydrogenase itself, but much shorter than would be expected from the molecular weights of the complexes. Models explaining the two lifetimes are discussed. A catalytic mechanism based on the internal mobility of the lipoamide dehydrogenase inside the multi-enzyme complex is proposed.A number of fluorescence studies on flavinadenine dinucleotide either free in solution or bound to a protein have been reported [I -61. Changes in fluorescence properties were used to detect conformational changes. Energy transfer studies to and from the flavin in lipoamide dehydrogenase and in the pyruvate dehydrogenase complex have been applied to obtain distance information in proteins and protein complexes. Natural donors, like tryptophan [2] as well as covalently bound fluorescent labels [7-101, have been used for this purpose.Previous lifetime studies employing the singlephoton counting technique on lipoamide dehydrogenase from pig heart by Visser et al. [ I l l revealed that the fluorescence decay of the bound flavins was consistent with the presence of two lifetimes. This result was confirmed by phase fluorometer data showing non-identical apparent lifetimes from phase and modulation [12]. Since lipoamide dehydrogenase is an enzyme consisting of two identical peptide chains and since the amplitudes as determined by single-~~ Ahhreviutzon TPP, thiamin diphosphate.photon counting were about identical, it was proposed that the two monomers have bound their respective FAD in a slightly different manner.The presence of two lifetimes is not only interesting, but also complicates considerably the conclusions from, for example, energy transfer. For this reason phase and modulation lifetimes of FAD in lipoamide dehydrogenase free and bound to pyruvate dehydrogenase complexes from other sources were compared [9,12]. The FAD in pyruvate dehydrogenase complex isolated from pig heart appeared to show even bigger differences in phase and modulation lifetimes...
We have attached eosin maleimide specifically to the lipoyl group of the pyruvate dehydrogenase complex isolated from Esclzerichia coli. Using this as the fluorescence acceptor and the intrinsic FAD of the lipoamide dehydrogenase subunit as the fluorescence donor, we confirmed previous measurements with other probes, in which it was suggested that the flavin moiety is at a substantial distance (over 4.5 nm) from the labeled lipoyl group. Since the lipoyl group must supply electrons to the FAD during the catalytic decarboxylation of pyruvate, we have investigated several potential mechanisms whereby this could happen. Movement within the complex, possibly triggered by the presence of substrate, seemed to be a strong possibility. Complex labeled with fluorophores on the accessible sulfhydryls, or on the lipoyl functions, did not give evidence of such triggering upon addition of substrate as judged by both static and dynamic fluorescence depolarization.The mobility of the subunits of labeled lipoamide dehydrogenase exceeded that expected for the total complex. Pyrene maleimide bound to the lipoyl functions also exhibited considerably faster rotations than the predicted one of the whole complex (z, > 3 ps). This suggests that a constant movement within the complex, coupled with the rotation of the lipoyl group, may bring the active sites of the complex transiently close enough together to interact on a time scale much faster than enzyme turnover. At the same time, the lipoyl group and the active sites of the complex can spend most of their time at points which are rather distant from each other.The pyruvate dehydrogenase complex from Eschericliia coli is a macromolecule with a molecular weight of at least 4.8 x lo6 [1,2] consisting of three enzymes (pyruvate dehydrogenase, lipoyl transacetylase and lipoamide dehydrogenase) which act in concert to cariry out the reactionThe pyruvate dehydrogenase and lipoamide dehydrogenase are noncovalently attached to the lipoyl transacetylase moiety which forms the core of the complex. The latter consists of 24 polypeptide subuni1.s each of which has two cofactor lipoic acid residues covalently bound by a lysyl amide bond. These lipolyl residues have been proposed to act as a 1.4-nmlong 'swinging arm' which passively transports acetyl enzyme^ The pyruvate dehydrogenase complex consists of pyruvate dehydrogenase, El (EC 1 2 4 l ) , lipoyl transacetylase, Ez (EC 2 3 1 12), and lipoamide dehydrogenase, E3 (EC 1 6 4 3 ) ~_ _ _ groups and electrons between the various subunit enzymes [ 3 ] . Recent evidence using fluorescence energy transfer techniques suggests, however, that the lipoyl groups when labeled with a fluorescent maleimide are at too great a distance (over 4.5 nm) from the FAD of the lipoamide dehydrogenase enzyme for the two to swing together in such a passive fashion [4-81. Similar measurements also place the lipoyl group at an equally long distance from the other active sites of the complex [9, lo].To date, all of the measurements of the distances between the flavin and ...
1. Purified pyridine nucleotide transhydrogenase from Azotobacter vinelandii contains three thiol residues as judged by titration with 5,5'-dithiobis(2-nitrobenzoic acid) under denaturing conditions.2. In the native conformation of the transhydrogenase only a single thiol residue is titrated. Modification of this exposed thiol does not influence transhydrogenase activity.3. The two less exposed thiol residues can be reacted in part with either p-chloromercuribenzoate or N-ethylnmleimide. Modification of one residue leads to loss of 40-60x of the enzyme activity in both the forward (NAD' + NADPH -+ NADH + NADP') and reverse reaction. The strong inhibitory action of phosphate ions on the reverse reaction Eur. J . Biochem. 107, is abolished after treatment with p-chloromercuribenzoate. Reaction with phenylmercurichloride or p-aminophenylmercuriacetate causes a similar activity loss without affecting the inhibitory action of phosphate.4. The interaction of the divalent thiol inhibitor p-aminophenylarsenoxide with transhydrogenase was found to be reversible and is characterized by an association constant of 6.3 x lo5 M-' at 25 "C in 50 mM sodium phosphate pH 7.50. This reversibility indicates formation of a cyclic dithiolarsinite derivative with considerable ring strain. The activity ofp-aminophenylarsenoxide-transhydrogenase is modulated by phosphate and magnesium ions. The activity of the transhydrogenase . p-aminophenylarsenoxide complex in the forward reaction is inhibited by phosphate and stimulated by magnesium ions. The reverse reaction is not catalyzed by the enzyme-inhibitor complex.5. The presence of an activity modulating site in transhydrogenase which binds phosphate ions and has the two less exposed thiol residues in close proximity is indicated by the results.Pyridine nucleotide transhydrogenase is potentially a very important enzyme in the bacterium Azotohucter vinelandii since it catalyzes the transfer of reduction equivalents from the NADP pool to the NAD pool or vice versa. Since the equilibrium constant of the reaction NADPH + NAD' +NADH + NADP' is approximately equal to unity [I], it follows that the enzyme will cease to catalyze a net transfer when the degrees of reduction of the two pools are approximately equal. Since a lot of NADPH is generated in the catabolism of A . vinelundii [2], the steady-state degree of reduction of the NADP pool is likely to be higher than that of the NAD pool. This is certainly the case in systems containing a membrane-bound, energy-linked transhydrogenase, e.g. mitochondria and Escherichia coli [3,4]. The normal direction of transfer of reduction equivalents in A . vinelandii catalyzed by transhydrogenase is thus likely to be from the ~ Abbrrviulions. sNAD', thionicotinamide adenine dinucleotide, oxidized form; NADPH + sNAD+, shorthand notation for the reaction NADPH + sNAD+ + NADP' + sNADH; CIHgBzOH, p-chloromercuribenzoate; PhHgC1, phenylmercurichloride; HzNPhHgOAc, p-aminophenylmercuriacetate; Nbsz, 5:5'-dithiobis(2-nitrobenzoic acid); H2NPh-AsO, p-aminophenylar...
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