Sulfur Clusters in LAM

Improvements in the procedure for purifying LAM, which included a more stringent adherence to anaerobicity, led to increased amounts of protein-bound iron, and allowed characterization of its configuration.LAM that is purified in an anaerobic chamber under conditions of less than 1 ppm of oxygen, normally contains 12-15 mol iron/mol hexamers, and exhibits a specific activity of 35-40 lU/mg, the maximum reported to date. The optical spectrum of the as-purified enzyme (Fig. 7) displays absorption features at 280 and 420 nm, a shoulder at 330 nm, and an additional broad feature that extends beyond 600 nm. These features indicate the presence of prosthetic groups in addition to pyridoxal 5 -phosphate, and are consistent with 

Electron paramagnetic resonance spectroscopy has contributed greatly to the understanding of the configuration of the iron in LAM. In initial spectroscopic studies, the as-purified enzyme exhibited a rhombic spectrum at 10 K. The signal was centered at g = 2.007, and underwent power saturation at the same temperature in a homogeneous manner with a = 15 2 mW. At higher temperatures the 

Inasmuch as this is not a defining characteristic of FcjSj clusters/ experiments were carried out to verify that the signal did not arise from either of these two reduced clusters. Treatment of the enzyme with 10 mM sodium dithionite in the presence of 6 jM methyl viologen as an oxidation-reduction mediator, resulted in complete loss of the as-isolated signal. This suggests that these clusters are subject to reduction rather than oxidation. The as-purified spectrum has therefore been tentatively attributed to Fe4S4 centers in the 3+ oxidation state. 

Despite the similarities in the spectra of the reduced state of LAM with the true cofactor and the cofactor analogue, there are nevertheless 

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Figure 10 Interaction of AdoMet with the iron sulfur cluster in LAM to generate the intermediate...

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