Hydrogenases use complex metal cofactors to catalyze the reversible formation of hydrogen. In [FeFe]-hydrogenases, the H-cluster cofactor includes a diiron subcluster containing azadithiolate, three CO, and two CN − ligands. During the assembly of the H cluster, the radical S-adenosyl methionine (SAM) enzyme HydG lyses the substrate tyrosine to yield the diatomic ligands. These diatomic products form an enzyme-bound Fe(CO) x (CN) y synthon that serves as a precursor for eventual H-cluster assembly. To further elucidate the mechanism of this complex reaction, we report the crystal structure and EPR analysis of HydG. At one end of the HydG (βα) 8 triosephosphate isomerase (TIM) barrel, a canonical [4Fe-4S] cluster binds SAM in close proximity to the proposed tyrosine binding site. At the opposite end of the active-site cavity, the structure reveals the auxiliary Fe-S cluster in two states: one monomer contains a [4Fe-5S] cluster, and the other monomer contains a [5Fe-5S] cluster consisting of a [4Fe-4S] cubane bridged by a μ 2 -sulfide ion to a mononuclear Fe 2+ center. This fifth iron is held in place by a single highly conserved protein-derived ligand: histidine 265. EPR analysis confirms the presence of the [5Fe-5S] cluster, which on incubation with cyanide, undergoes loss of the labile iron to yield a [4Fe-4S] cluster. We hypothesize that the labile iron of the [5Fe-5S] cluster is the site of Fe (CO) x (CN) y synthon formation and that the limited bonding between this iron and HydG may facilitate transfer of the intact synthon to its cognate acceptor for subsequent H-cluster assembly.radical SAM enzyme | tyrosine lyase | H-cluster biosynthesis T he assembly of the [FeFe]-hydrogenase diiron subcluster (1, 2) requires three maturase proteins, HydE, HydF, and HydG (3), and in vitro, they can assemble an active hydrogenase (4). The sequence and structure of the maturase HydE (5) indicates that it is a member of the radical S-adenosyl methionine (SAM) superfamily, although the biochemical function of HydE has not been experimentally determined. The GTPase HydF (6, 7) has been shown to transfer synthetic (8) or biologically derived (7, 9) diiron subclusters into apo-hydrogenase, suggesting that HydF functions as a template for diiron subcluster assembly. The tyrosine lyase HydG is also a member of the radical SAM superfamily and uses SAM and a reductant (such as dithionite) to cleave the Cα-Cβ bond of tyrosine, yielding p-cresol as the side chain-derived byproduct (10) and fragmenting the amino acid moiety into cyanide (CN − ) (11) and carbon monoxide (CO) (12), which are ultimately incorporated as ligands in the H cluster of the [FeFe]-hydrogenase HydA (4). Two site-differentiated [4Fe-4S] clusters in HydG have been identified using a combination of spectroscopy and site-directed mutagenesis (12-16). The cluster bound close to the N terminus ([4Fe-4S] RS ) by the CX 3 CX 2 C cysteine triad motif (SI Appendix, Fig. S1) is typical of the radical SAM superfamily (17, 18) and has been shown to catalyze the reductive cl...

Summary Statement: The biosynthesis of lipoyl cofactors requires two lipoyl synthase mediated sulfur insertions. We report the crystal structures of a lipoyl synthase complexed with S-adenosylhomocysteine or 5'-methylthioadenosine. Models based on these structures identify likely substrate binding sites.Keywords: radical SAM, cofactors, crystal structure, enzyme catalysis, sulfur Abbreviations used: LipA, lipoyl synthase; BioB, biotin synthase; SAM, Sadenosylmethionine; LCD, lipoyl carrier domain; MTA, 5'-methylthioadenosine; RS, radical SAM; ACP, acyl carrier protein; SsLipA, Sulfolobus solfataricus LipA; TeLipA2 Thermosynechococcus elongatus LipA2; Ec, Escherichia coli; 5'-dA, 5'-deoxyadenosine. 2 ABSTRACTLipoyl cofactors are essential for living organisms and are produced by the insertion of two sulfur atoms into the relatively unreactive C-H bonds of an octanoyl substrate. This reaction requires lipoyl synthase, a member of the radical SAM enzyme superfamily. Herein we present crystal structures of lipoyl synthase with two [4Fe-4S] clusters bound at opposite ends of the TIM barrel, the usual fold of the radical SAM superfamily. The cluster required for reductive SAM cleavage conserves the features of the radical SAM superfamily, but the auxiliary cluster is bound by a CX 4 CX 5 C motif unique to lipoyl synthase. The fourth ligand to the auxiliary cluster is an extremely unusual serine residue. Site directed mutants show this conserved serine ligand is essential for the sulfur insertion steps. One crystallized LipA complex contains MTA, a breakdown product of SAM, bound in the likely SAM binding site. Modelling has identified an 18 Å deep channel, well-proportioned to accommodate an octanoyl substrate. These results suggest the auxiliary cluster is the likely sulfur donor, but access to a sulfide ion for the second sulfur insertion reaction requires the loss of an iron atom from the auxiliary cluster, which the serine ligand may enabled.3

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Recently, variants of implicit solvation models for first principles electronic structure calculations based on a direct solution of the nonhomogeneous Poisson equation in real space have been developed. These implicit solvation models are very elegant from a physical point of view as the solute cavity is defined directly via isosurfaces of the electronic density, and the molecular charge is polarized self‐consistently by the reaction field of the dielectric continuum which surrounds the solute. Nevertheless, the implementation of these models is technically complex and requires great care. A certain level of care is required from users of such models as a number of numerical parameters need to be given appropriate values to obtain the most accurate and physically relevant results. Here, we describe in what parts of the solvent model each of these numerical parameters is involved and present a detailed study of how they can affect the calculation, using the solvation model which has been implemented in the ONETEP program for linear‐scaling density functional theory (DFT) calculations. As ONETEP is capable of DFT calculations with thousands of atoms, we focus our investigation of the numerical parameters with a case study on protein–ligand complexes of the entire 2602‐atom T4 Lysozyme L99/M102Q protein. We examine effects on solvation energies and binding energies, which are critical quantities for computational drug optimization and other types of biomolecular simulations. We propose optimal choices of these parameters suitable for routine “production” calculations. © 2012 Wiley Periodicals, Inc.

Schemes of increasing sophistication for obtaining free energies of binding have been developed over the years, where configurational sampling is used to include the all-important entropic contributions to the free energies. However, the quality of the results will also depend on the accuracy with which the intermolecular interactions are computed at each molecular configuration. In this context, the energy change associated with the rearrangement of electrons (electronic polarization and charge transfer) upon binding is a very important effect. Classical molecular mechanics force fields do not take this effect into account explicitly, and polarizable force fields and semiempirical quantum or hybrid quantum-classical (QM/MM) calculations are increasingly employed (at higher computational cost) to compute intermolecular interactions in free-energy schemes. In this work, we investigate the use of large-scale quantum mechanical calculations from first-principles as a way of fully taking into account electronic effects in free-energy calculations. We employ a one-step free-energy perturbation (FEP) scheme from a molecular mechanical (MM) potential to a quantum mechanical (QM) potential as a correction to thermodynamic integration calculations within the MM potential. We use this approach to calculate relative free energies of hydration of small aromatic molecules. Our quantum calculations are performed on multiple configurations from classical molecular dynamics simulations. The quantum energy of each configuration is obtained from density functional theory calculations with a near-complete psinc basis set on over 600 atoms using the ONETEP program.