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-H-2]-NADH was studied anaerobically at 4 degreesC 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)) similar to 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, EH2. 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, EH4. 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 EH2 or slow release of NAD(+) before rapid NADH binding and reaction to form EH4. The mechanism of oxidation of EH2 by NAD(+) was also investigated under the same conditions. The 530 nm charge-transfer absorbance of EH2 shifted to 600 nm upon NAD(+) binding in the dead time of mixing of the stopped-flow instrument and represents formation of the EH2.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 similar to115 s(-1) probably represents the overall rate of formation of E-ox and NADH from EH2 and NAD(+), and is largely determined by the slower rates of the coupled sequence of reactions preceding flavin oxidation.