Abstract
Maturation of the [FeFe]-hydrogenase active site depends on at least the expression of three gene products called HydE, HydF, and HydG. We have solved the high resolution structure of recombinant, reconstituted S-adenosine-L-methionine-dependent HydE from Thermotoga maritima. Besides the conserved [Fe(4)S(4)] cluster involved in the radical-based reaction, this HydE was reported to have a second [Fe(4)S(4)] cluster coordinated by three Cys residues. However, in our crystals, depending on the reconstitution and soaking conditions, this second cluster is either a [Fe(2)S(2)] center, with water occupying the fourth ligand site or is absent. We have carried out site-directed mutagenesis studies on the related HydE from Clostridium acetobutylicum, along with in silico docking and crystal soaking experiments, to define the active site region and three anion-binding sites inside a large, positive cavity, one of which binds SCN(-) with high affinity. Although the overall triose-phosphate isomerase-barrel structure of HydE is very similar to that of biotin synthase, the residues that line the internal cavity are significantly different in the two enzymes.
Highlights
Requirement of maturation machineries in order to obtain active enzymes for each of the three classes
Comparison with Biotin Synthase and Other AdoMet Radical Proteins—Overall, HydE is significantly similar to E. coli BioB; the superposition of both enzymes gives a root mean square deviation of 2.5 Å for 292 of 348 C␣ atoms
The two additional helices contact the triose-phosphate isomerase (TIM)-barrel helix 6 that in both proteins is composed of several invariant hydrophobic side chains and either one arginine or lysine residue [19]
Summary
18862 JOURNAL OF BIOLOGICAL CHEMISTRY nate, cyanate, cyanide, carbamoylphosphate, phosphate, cysteine, pyruvate, asparagine, succinamate, maleamate, glycine, acetate, and lactate These molecules were considered to be either plausible substrates, products, precursors, or their fragments. Anomalous Scattering Labeling—In order to probe the putative anion-binding sites, crystals were flash-cooled within 5 min from a solution containing 100 mM NaBr. X-ray diffraction data were subsequently collected at a slightly higher energy than the absorption edge for bromine. X-ray diffraction data were subsequently collected at a slightly higher energy than the absorption edge for bromine In another experiment, the protein was co-crystallized with NaI, and the crystal was flashcooled within 5 min in a cryoprotecting solution containing NaCl. The fraction of remaining bound IϪ was determined by measuring the residual x-ray anomalous signal at ϭ 0.933 Å. The less occupied side chain alternative positions as well as all of the ions, detergent, and water molecules were deleted from the file
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