Crystal Structure of the [FeFe]-Hydrogenase Maturase HydE Bound to Complex-B

Abstract

[FeFe]-hydrogenases use a unique organometallic complex, termed the H cluster, to reversibly convert H2 into protons and low-potential electrons. It can be best described as a [Fe4S4] cluster coupled to a unique [2Fe]H center where the reaction actually takes place. The latter corresponds to two iron atoms, each of which is bound by one CN– ligand and one CO ligand. The two iron atoms are connected by a unique azadithiolate molecule (−S–CH2–NH–CH2–S–) and an additional bridging CO. This [2Fe]H center is built stepwise thanks to the well-orchestrated action of maturating enzymes that belong to the Hyd machinery. Among them, HydG converts l-tyrosine into CO and CN– to produce a unique l-cysteine-Fe­(CO)2CN species termed complex-B. Very recently, HydE was shown to perform radical-based chemistry using synthetic complex-B as a substrate. Here we report the high-resolution crystal structure that establishes the identity of the complex-B-bound HydE. By triggering the reaction prior to crystallization, we trapped a new five-coordinate Fe species, supporting the proposal that HydE performs complex modifications of complex-B to produce a monomeric “SFe­(CO)2CN” precursor to the [2Fe]H center. Substrate access, product release, and intermediate transfer are also discussed.

L-tyrosine-bound ThiH structure reveals C–C bond break differences within radical SAM aromatic amino acid lyases

“…HydG shares this catalytic step in accordance with their active site homology. Furthermore, our modeling of the 5´-dA• hydrogen abstraction in ThiH and NosL reveals that the compactness of the site brings 5´-dA• very close to the amino group, inducing hydrogen tunneling for both enzymes; this can probably be generalized to all radical SAM enzymes given the similar compactness also observed in available [5´-dA]–substrate bound structures 45 , 46 and in [SAM]-substrate bound structures 13 , 47 . It also shows that, despite exhibiting conserved active sites, the sum of tiny structural differences is likely to be responsible for subtle changes during this step that will orient the reaction toward the subsequent Cα–Cβ or the Cα–C bond break in ThiH / HydG and NosL, respectively.…”

Quantum Chemical Study of a Radical Relay Mechanism for the HydG-Catalyzed Synthesis of a Fe(II)(CO)2(CN)cysteine Precursor to the H-Cluster of [FeFe] Hydrogenase

“…This computed mechanism produces an isomer of the synthon where the CN ligand is opposite to the cysteinyl carboxylate oxygen, whereas a recent experimental study on the crystal structure of HydE with the synthon as its substrate assigned the CN ligand as opposite to sulfur based on the proximity of hydrogen bond-donating residues. We computed the relative electronic energies of the synthon isomers and found them to be very close in energy (Δ E (opposite S) = 0.00; Δ E (opp.…”

Proposed Mechanism for the Biosynthesis of the [FeFe] Hydrogenase H-Cluster: Central Roles for the Radical SAM Enzymes HydG and HydE

“…Figure 7 summarizes recent Thermotoga maritima TmHydE crystal structures obtained by crystallizing this rSAM enzyme with the synthon carrier Syn-B. 54 Figure 7A zooms in on the substrate binding site as found in the "upper part" of HydE's β barrel cavity. For this component of the HydE crystal stucture study, focusing on the substrate binding site, the nonreactive SAM analogue (S)-adenosyl-L-homocysteine (SAH) was used in place of SAM.…”

“…Tao et al showed with EPR spectroscopy that the radical SAM reaction of HydE indeed catalyzes the conversion of the HydG produced synthon, initially forming the adenosylated Fe­(I) intermediate via a the 5′dAdo • attack on the cysteine sulfur of the synthon. This spectroscopic study was soon followed by an X-ray crystallography study by Rohac et al showing the specific binding site of the synthon adjacent to the rSAM [4Fe–4S] cluster.…”

“…Figure 7. Summary of crystal structures of Thermotoga maritima T m H y d E i n t e r a c t i n g w i t h S y n -B -d o n a t e d [ F e ( C N ) -(CO) 2 (cysteinate)] − , from PDB entry 701T 54. …”