Destruction and reformation of an iron-sulfur cluster during catalysis by lipoyl synthase

Abstract

Lipoyl synthase (LipA) catalyzes the last step in the biosynthesis of the lipoyl cofactor, which is the attachment of two sulfhydryl groups to C6 and C8 of a pendant octanoyl chain. The appended sulfur atoms derive from an auxiliary [4Fe-4S] cluster on the protein that is degraded during turnover, limiting LipA to one turnover in vitro. We found that the Escherichia coli iron-sulfur (Fe-S) cluster carrier protein NfuA efficiently reconstitutes the auxiliary cluster during LipA catalysis in a step that is not rate-limiting. We also found evidence for a second pathway for cluster regeneration involving the E. coli protein IscU. These results show that enzymes that degrade their Fe-S clusters as a sulfur source can nonetheless act catalytically. Our results also explain why patients with NFU1 gene deletions exhibit phenotypes that are indicative of lipoyl cofactor deficiencies.

Fig. 1

Catalysis by LipA. LipA contains two [4Fe–4S] clusters, one of which (the auxiliary cluster shown) is sacrificed during turnover. Catalysis proceeds by reductive cleavage of SAM to render a 5′-deoxyadenosyl 5′-radical (5′-dA•), which abstracts the C6 pro-R hydrogen atom of a pendant n-octanoamide chain residing on a lipoyl carrier protein (H protein of the glycine cleavage system shown). The resulting C6 substrate radical attacks one of the sulfide ions of the auxiliary cluster, which is followed by loss of an Fe²⁺ ion to afford a [3Fe–3S–1(6S)-thio-octanoamide]H protein intermediate (labeled as monothiolated octanoyl H protein). A second reductive cleavage of SAM generates a second 5′-dA•, which abstracts an H• from C8 of the thio-octanoamide H protein intermediate. The resulting C8 substrate radical attacks a second sulfide ion of the auxiliary cluster, which is followed by the addition of two protons and the loss of three Fe²⁺ ions and two S⁻ ions to generate the lipoyl group in its reduced form.

Fig. 2

(A) UV-visible spectrum of 15 μM as-isolated E. coli NfuA. (B) Interaction between LipA and NfuA monitored by molecular sievechromatography. Dashed line, 100 μM NfuA alone. NfuA elutes (64.6 mL) with an experimentally calculated molecular mass of 28.9 kDa (theoretical mass, 25.6 kDa). Dotted line, 100 μM LipA alone. LipA elutes (59.6 mL) with an experimentally calculated molecular mass of 44.1 kDa (theoretical mass, 38.2 kDa). Solid line, 100 μM LipA + 100 μM NfuA. A complex between LipA and NfuA elutes (54.6 mL) with an experimentally calculated molecular mass of 67.2 kDa (theoretical mass, 63.8 kDa). (C) SDS–PAGE gel of fraction represented by solid line in panel B (lane 2). NfuA standard (lane 3). Molecular mass markers (lane 1). (D) Addition of NfuA into a LipA reaction gives rise to additional turnovers. 6(S)-thio-octanoyllysyl intermediate (closed triangles). Lipoyllysyl product (closed squares). The reaction contained 50 μM LipA, 600 μM peptide substrate analog (Glu-Ser-Val-[N⁶-octanoyl]Lys-Ala-Ala-Ser-Asp), 0.5 μM SAH nucleosidase, 2 mM dithionite and 1 mM SAM. Reactions were conducted at room temperature, and at the designated time indicated by the arrow, 200 μM NfuA was added to the reaction (E) Inclusion of NfuA (200 μM) in LipA reaction mixtures gives rise to multiple turnovers. Lipoyllysyl product in the absence of NfuA (closed squares). Lipoyllysyl product in the presence of NfuA (closed triangles). The reaction conditions were as described for panel D. (F) The LipA reaction under catalytic conditions. The reaction conditions were as described in panel A, except that the concentration of LipA was 10 μM (7 μM active) and the concentration of NfuA was 400 μM.

Fig. 3

(A) Formation of lipoyllysyl product in the presence of NfuA reconstituted with ³⁴S-labeled sulfide. (³²S,³²S)-containing lipoyllysyl product (closed circles); (³⁴S,³⁴S)-containing lipoyllysyl product (closed triangles); (³²S,³⁴S)-containing lipoyllysyl product (closed squares); total lipoyllysyl product (open squares). The reaction contained 20 μM LipA, 100 μM ³⁴S-labeled NfuA, 600 μM peptide substrate analog, 0.5 μM SAH nucleosidase, 2 mM dithionite and 1 mM SAM. (B) Formation of lipoyllysyl product in the presence of NfuA reconstituted with ³⁴S-labeled sulfide and under catalytic conditions. The reaction conditions were as described above, except that the concentration of LipA was 10 μM and the concentration of ³⁴S-labeled NfuA was 400 μM. (³²S,³²S)-containing lipoyllysyl product (closed circles); (³⁴S,³⁴S)-containing lipoyllysyl product (closed triangles). (C) Effect of extraneous iron and sulfide on the activity of LipA. LipA reaction (50 μM) in the absence of NfuA or iron and sulfide (closed squares). LipA reaction in the presence of 0.8 mM FeCl₃ and 0.8 mM Na₂S (open squares). LipA reaction in the presence of 200 μM NfuA (closed triangles). Other reaction components were as described above in panel A. (D) Effect of IscS on the LipA reaction. LipA reaction in the presence of IscS (open triangles). LipA reaction in the absence of IscS (closed squares). Reactions were described as above (panel A), except they contained 100 μM LipA, 200 μM IscS, 5 mM cysteine, 5 mM DTT, and no NfuA. (E) Effect of citrate on NfuA’s enhancement of LipA catalysis. Reactions included 100 μM LipA (open squares); 100 μM LipA + 5 mM citrate (closed squares); 100 μM LipA + 1 mM NfuA (open circles); 100 μM LipA + 1 mM NfuA + 5 mM citrate (closed circles). (F) Reaction of 25 μM LipA + 400 μM NfuA (closed squares); 25 μM LipA, and 400 μM NfuA in the presence of 1 mM Na₂³⁴S (closed triangles) in which the lipoic acid product containing two ³²S atoms is monitored. Reaction of 25 μM LipA + 400 μM NfuA in the presence of 1 mM Na₂³⁴S monitoring the ³²S/³⁴S mixed-labeled product (closed circles) and the ³⁴S/³⁴S-labeled product (open circles).

Fig. 4

(A) Effect of NfuA on growth of E. coli in M9 minimal medium using glucose as a carbon source. Wild-type BW25113 (closed circles), BW25113:ΔnfuA (solid squares), BW25113:ΔnfuA + 25 μM lipoic acid (open circles), and BW25113:ΔnfuA + 5 mM succinate/5 mM acetate (black triangles). (B) Effect of IscU on LipA catalysis. LipA only (solid squares). LipA + IscU (solid triangles). LipA + NfuA (solid circles). (C) Effect of IscU, HscA, and HscB on LipA catalysis. LipA only (solid squares). LipA + IscU (solid circles). LipA + IscU + HscA and HscB (solid triangles). (D) Effect of NfuA, HscA, and HscB on LipA catalysis. LipA only (solid squares). LipA + NfuA (solid circles). LipA + NfuA + HscA and HscB (solid triangles). Reactions contained 20 μM LipA, 400 μM octanoyllysyl-containing peptide substrate, 2 mM SAM, 2 mM dithionite, 2 mM ATP, 100 mM MgCl₂, and 0.5 μM SAH nucleosidase. When appropriate, IscU, HscA, and HscB were each added to a final concentration of 200 μM. (E) Reaction of 25 μM LipA + 400 μM IscU (closed squares); 25 μM LipA, and 400 μM IscUin the presence of 1 mM Na₂³⁴S (closedsquares) in which the lipoic acid product containing two ³²S atoms is monitored. Reaction of 25 μM LipA + 400 μM IscU in the presence of 1 mM Na₂³⁴S monitoring the ³²S/³⁴S mixed-labeled product (closed triangles) and the ³⁴S/³⁴S doubled-labeled product (open triangles).