Hydrogen Deuterium Exchange Mass Spectrometry Reveals Unique Conformational and Chemical Transformations Occurring upon 4Fe-4S Cluster Binding in the Type 2 L-Serine Dehydratase from Legionella pneumophila

摘要

The Type 2 L-serine dehydratase from Legionella pneumophila (lpLSD) contains a [4Fe-4S]²⁺ cluster that acts as a Lewis acid to extract the hydroxyl group of L-serine during the dehydration reaction. Surprisingly, the crystal structure showed that all four of the iron atoms in the cluster were coordinated with protein cysteinyl residues and that the cluster was buried and not exposed to solvent. If the crystal structure of lpLSD accurately reflects the structure in solution, then substantial rearrangement at the active site is necessary for the substrate to enter. Furthermore, repair of the oxidized protein when the cluster has degraded would presumably entail exposure of the buried cysteine ligands. Thus, the conformation required for substrate to enter may be similar to those required for a new cluster to enter the active site. To address this, hydrogen-deuterium exchange combined with mass spectrometry (HDX MS) was used to probe the conformational changes that occur upon oxidative degradation of the Fe-S cluster. The regions that show the most significant differential HDX are either adjacent to the cluster location in the holo enzyme or connect regions that are adjacent to the cluster. The observed decrease in flexibility upon cluster binding provides direct evidence that the “tail in mouth” conformation observed in the crystal structure also occurs in solution and that the C-terminal peptide is coordinated to the [4Fe-4S] cluster in a pre-catalytic conformation. This observation is consistent with the requirement of an activation step prior to catalysis and with the unusually high level of resistance to oxygen-induced cluster degradation. Furthermore, peptide mapping of the apo form under non-reducing conditions revealed the formation of disulfide bonds between C396 and C485 and possibly between C343 and C385. These observations provide a picture of how the cluster loci are stabilized and poised to receive the cluster in the apo form and the requirement for an oxidation step in cluster formation.