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. 2024 Feb 6;14(1):3026.
doi: 10.1038/s41598-024-53745-2.

Evidence the Isc iron-sulfur cluster biogenesis machinery is the source of iron for [NiFe]-cofactor biosynthesis in Escherichia coli

Alexander Haase #  1 , Christian Arlt #  2 , Andrea Sinz  2 , R Gary Sawers  3
Affiliations

Evidence the Isc iron-sulfur cluster biogenesis machinery is the source of iron for [NiFe]-cofactor biosynthesis in Escherichia coli

Alexander Haase et al. Sci Rep. .

Abstract

[NiFe]-hydrogenases have a bimetallic NiFe(CN)2CO cofactor in their large, catalytic subunit. The 136 Da Fe(CN)2CO group of this cofactor is preassembled on a distinct HypC-HypD scaffold complex, but the intracellular source of the iron ion is unresolved. Native mass spectrometric analysis of HypCD complexes defined the [4Fe-4S] cluster associated with HypD and identified + 26 to 28 Da and + 136 Da modifications specifically associated with HypC. A HypCC2A variant without the essential conserved N-terminal cysteine residue dissociated from its complex with native HypD lacked all modifications. Native HypC dissociated from HypCD complexes isolated from Escherichia coli strains deleted for the iscS or iscU genes, encoding core components of the Isc iron-sulfur cluster biogenesis machinery, specifically lacked the + 136 Da modification, but this was retained on HypC from suf mutants. The presence or absence of the + 136 Da modification on the HypCD complex correlated with the hydrogenase enzyme activity profiles of the respective mutant strains. Notably, the [4Fe-4S] cluster on HypD was identified in all HypCD complexes analyzed. These results suggest that the iron of the Fe(CN)2CO group on HypCD derives from the Isc machinery, while either the Isc or the Suf machinery can deliver the [4Fe-4S] cluster to HypD.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Effects of mutations in key genes of iron metabolism on hydrogenase enzyme activity. (a) In-gel hydrogenase enzyme activity determined in extracts of the indicated strains. Aliquots (25 μg of protein) of crude extracts were separated in clear-native polyacrylamide (7.5% w/v) gels. (b) Aliquots (25 μg of protein) of the same crude extracts shown in part (a) were separated under denaturing conditions in SDS-PAGE (12.5% w/v acrylamide). After transfer to a nitrocellulose membrane, the HypD polypeptide was identified (see arrow) using antiserum raised against HypD (dilution 1:4000). The asterisk denotes an unidentified cross-reacting polypeptide. (c) As in part (b) but stained with Coomassie-Brilliant-Blue. Molecular mass markers are shown in kDa on the left of panels (b,c).
Figure 2
Figure 2
Plasmid-encoded native HypCD complex fails to restore H2 evolution to E. coli isc mutants. Total hydrogen gas accumulated in the headspace of anaerobic cultures of the indicated strains, with or without plasmid pT-hypDCStrep, and grown for 22 h at 37 °C in TGYEP medium is shown.
Figure 3
Figure 3
Purified StrepII-tagged HypCD complexes isolated from strains with defects in iron metabolism. (a) Coomassie-Brilliant-Blue-stained SDS-PAGE (12.5% w/v polyacrylamide) of purified StrepII-tagged HypCD complexes (5 μg protein) isolated from the indicated strains. (b) A gel identical to that shown in part a), but challenged with antiserum containing antibodies specific for HypD (diluted 1:4000). (c) A gel similar to that shown in part (a), but with 7.5 μg of protein applied per lane and challenged with antiserum containing antibodies against HypC (diluted 1:4000). Molecular mass markers in kDa are shown on the left of each panel. Arrows identify the migration positions of HypD or HypC.
Figure 4
Figure 4
UV–Vis spectroscopy indicates HypD contains a [4Fe–4S] cluster. Shown are UV–Vis spectra of StrepII-tagged HypCD complexes (1 mg ml−1) isolated from the strains indicated. (a) HypCD isolated from DHP-D (∆hypD) transformed with pT-hypDCStrep (black spectrum) or from CP1244 (∆iscU) transformed with pT-hypDCStrep (gray spectrum); (b) HypCD isolated from CP1233 (∆sufA) (black spectrum) transformed with pT-hypDCStrep, or CP742 (∆iscA-∆erpA) transformed with pT-hypDCStrep (light blue spectrum).
Figure 5
Figure 5
Native mass spectra of HypCD complexes isolated from different E. coli mutants. (a) Native mass spectrum of native HypCD complex (upper panel) and HypCC2AD complex (lower panel) isolated from strain DHP-D (∆hypD) transformed with pT-hypDCStrep or pT-hypDC(C2A)Strep, respectively, and analysed at a collision energy of 30 V. Stoichiometry of the main complex species is StrepII-HypC:HypD of 1:1 (indicated by red circles for HypC and green circles for HypD), with minor species showing 2:1 (#) and 2:2 (*) ratios. (b) Native mass spectra of the HypCD complex isolated from strains CP1233 (∆sufA), CP1244 (∆iscU) or CP411 (∆entC-∆feoB). All strains carried plasmid pT-hypDCStrep. Signals corresponding to HypC (red overlay), HypD (green overlay) and the 1:1 complex (red-green overlay) are labeled accordingly.
Figure 6
Figure 6
Native MS spectra of HypC and HypD dissociated from HypCD complexes reveal absence of the + 136 Da modification on HypC in isc mutants but presence of the [4Fe–4S] cluster on HypD. (a) Mass spectrum of the dissociation of the + 12 charged ion species of the HypCD heterodimer into HypC (charge states + 4 through + 6, red spheres) and HypD (charge states + 6 through + 8, green spheres) at a collision energy of 90 V isolated from strain DHP-D transformed with plasmid pT-hypDCStrep as positive control. (b) Zoom in for HypC (charge state + 5), as well as (c) zoom in for HypD (charge state + 7) are shown for isolated complexes from strains transformed with pT-hypDCStrep: DHP-D (“WT”), CP1233 (∆sufA), CP1244 (∆iscU) and CP411 (∆entC-feoB), and HypCC2A dissociated from the HypCC2AHypD complex isolated from strain DHP-D (∆hypD) transformed with pT-hypDC(C2A)Strep. Signals are labeled with the corresponding m/z value in the first zoomed row. Potential modifications are indicated above the colored overlay. Note that a putative methyl thiazolidine modification accounting for the + 26 Da species is not indicated in the Figure.
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