Abstract
Lipoyl synthase (LipA) catalyses the final step of the biosynthesis of the lipoyl cofactor by insertion of two sulfur atoms at the C6 and C8 atoms of the protein-bound octanoyl substrate. In this reaction, two [4Fe4S] clusters and two molecules of S-adenosyl-l-methionine are used. One of the two FeS clusters is responsible for the generation of a powerful oxidant, the 5′-deoxyadenosyl radical (5′-dA•). The other (the auxiliary cluster) is the source of both sulfur atoms that are inserted into the substrate. In this paper, the spin state of the FeS clusters and the reaction mechanism is investigated by the combined quantum mechanical and molecular mechanics approach. The calculations show that the ground state of the two FeS clusters, both in the [4Fe4S]2+ oxidation state, is a singlet state with antiferromagnetically coupled high-spin Fe ions and that there is quite a large variation of the energies of the various broken-symmetry states, up to 40 kJ/mol. For the two S-insertion reactions, the highest energy barrier is found for the hydrogen-atom abstraction from the octanoyl substrate by 5′-dA•. The formation of 5′-dA• is very facile for LipA, with an energy barrier of 6 kJ/mol for the first S-insertion reaction and without any barrier for the second S-insertion reaction. In addition, the first S ion attack on the C6 radical of octanoyl was found to take place directly by the transfer of the H6 from the substrate to 5′-dA•, whereas for the second S-insertion reaction, a C8 radical intermediate was formed with a rate-limiting barrier of 71 kJ/mol.
Highlights
Lipoic acid is a sulfur-containing cofactor that is essential for living organisms
The properties of [4Fe4S]2+ clusters have been studied extensively [17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33]. They can be affected by environment, e.g., different ligands binding to Fe ion or the protein surrounding
The quantum mechanical and molecular mechanics (QM/MM) approach was employed, taking into account the surrounding protein and solvent. Both clusters are in the [4Fe4S]2+ oxidation state, i.e., each with two reduced Fe2+ and two oxidised F e3+ ions. This gives a total of 4 × (4 + 5) = 36 unpaired spins in the high-spin state, which can be combined in many different ways
Summary
Lipoic acid is a sulfur-containing cofactor that is essential for living organisms. It is employed in acyl-transfer reactions in several enzymes, including the pyruvate and α-ketoglutarate dehydrogenases in the citric acid cycle [1]. Lipoyl synthase (LipA) is a metalloenzyme that catalyses the final step in the biosynthesis of this cofactor by the insertion of two sulfur atoms at the C6 and C8 atoms of the octanoyl substrate, attached to the lipoyl carrier protein (Scheme 1) [2,3,4,5]. In the resting state (without octanoyl substrate and AdoMet binding to active site), the auxiliary [4Fe4S] cluster is bound to the protein with one Ser and three Cys residues, whereas three Cys resides and one solvent molecule coordinate to the other cluster. During the first S-insertion reaction, the Ser residue dissociates from the auxiliary FeS cluster and one Fe ion is lost, whereas one of the sulfide ions
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