Archaeal ApbC/Nbp35 homologs function as iron-sulfur cluster carrier proteins

Abstract

Iron-sulfur clusters may have been the earliest catalytic cofactors on earth, and most modern organisms use them extensively. Although members of the Archaea produce numerous iron-sulfur proteins, the major cluster assembly proteins found in the Bacteria and Eukarya are not universally conserved in archaea. Free-living archaea do have homologs of the bacterial apbC and eukaryotic NBP35 genes that encode iron-sulfur cluster carrier proteins. This study exploits the genetic system of Salmonella enterica to examine the in vivo functionality of apbC/NBP35 homologs from three archaea: Methanococcus maripaludis, Methanocaldococcus jannaschii, and Sulfolobus solfataricus. All three archaeal homologs could correct the tricarballylate growth defect of an S. enterica apbC mutant. Additional genetic studies showed that the conserved Walker box serine and the Cys-X-X-Cys motif of the M. maripaludis MMP0704 protein were both required for function in vivo but that the amino-terminal ferredoxin domain was not. MMP0704 protein and an MMP0704 variant protein missing the N-terminal ferredoxin domain were purified, and the Fe-S clusters were chemically reconstituted. Both proteins bound equimolar concentrations of Fe and S and had UV-visible spectra similar to those of known [4Fe-4S] cluster-containing proteins. This family of dimeric iron-sulfur carrier proteins evolved before the archaeal and eukaryal lineages diverged, representing an ancient mode of cluster assembly.

FIG. 1.

A protein sequence alignment of bacterial, archaeal, and eukaryotic ApbC/Nbp35 homologs was constructed using the ClustalW program (version 1.83) (37). The sequence of the S. enterica serovar Typhimurium protein (ApbC; RefSeq accession no. NP_461098.1) is shown without the amino-terminal domain that is not homologous to the amino-terminal domains of the archaeal and eukaryotic proteins. The archaeal homologs are from S. solfataricus (SSO0460; accession no. NP_341994.1), P. furiosus (PF1145; accession no. NP_578874.1), Methanosarcina acetivorans (MA4246; accession no. NP_619111.1), M. jannaschii (MJ0283; accession no. NP_247256.1), and M. maripaludis (MMP0704; accession no. NP_987824.1). The two paralogs from S. cerevisiae are Nbp35 (accession no. NP_011424.1) and Cfd1 (accession no. NP_012263.1). Conserved amino acid residues are shown in white on a black background. Similar residues are shown in black on a gray background. The four conserved amino-terminal cysteine residues shared by the MMP0704 and Nbp35p proteins are boxed. Asterisks above the sequences indicate MMP0704 residues replaced by mutagenesis in this study. A vertical bar indicates the N termini of the truncated proteins MJ0283(19-290) and MMP0704(20-289).

FIG. 2.

The ApbC/Nbp35 family of iron-sulfur proteins evolved before the Archaea and Eukarya diverged. This phylogeny was inferred using a protein maximum-likelihood method, although a neighbor-joining distance method produced a similar tree. Bootstrap values from the neighbor-joining consensus tree are shown near branches supported by a plurality of 100 trees. Numbers in parentheses after some species names differentiate paralogs from the same genome. Asterisks indicate sequences with amino-terminal ferredoxin-like domains. The complete organism names and accession numbers are listed in the supplemental material. Bar, 0.1 amino acid substitution expected per site.

FIG. 3.

M. maripaludis MMP0704 constructs can complement an S. enterica apbC mutant. Strains were grown at 37°C in no-carbon essential salts medium supplemented with thiamine, with tricarballylate as the sole carbon and energy source. (A) The growth of strain DM10300 with pJMB100 (apbC) (open squares), pJMB105 (Δ1-19 MMP0704) (open circles), pJMB109 [MMP0704(Cys5,9,12,18Ala)] (filled triangles), pJMB104 (MMP0704) (filled circles), and empty vector (pSU18) (filled squares) was determined by measuring the OD₆₅₀. (B) Mutational analysis of conserved cysteine residues in the Δ1-19 MMP0704 gene product. The growth of strain DM10300 with pJMB105 (Δ1-19 MMP0704) (open circles), pJMB111 [Δ1-19 MMP0704(Cys218Ala)] (closed squares), pJMB114 [Δ1-19 MMP0704(Cys220Ala)] (open squares), and pJMB115 [Δ1-19 MMP0704(Cys218,220Ala)] (closed circles) was monitored as described in the legend to panel A.

FIG. 4.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of an overloaded, Coomassie blue-stained gel shows the purities and solubilities of heterologously expressed MMP0704 and MJ0283 proteins (10 μg each). Marker proteins, shown in lane M, have the molecular masses indicated to the left. Lane 1 contains affinity-purified MMP0704-His₆ protein, with an apparent molecular mass of 33 kDa (calculated mass, 32.1 kDa). Lane 2 contains affinity-purified MMP0704(20-289)-His₆ protein, with an apparent molecular mass of 29 kDa (calculated mass, 30.4 kDa). Lane 3 contains heat-stable, purified MJ0283 protein, with an apparent molecular mass of 31 kDa (calculated mass, 31.2 kDa). Lane 4 contains heat-stable, purified MJ0283(19-290) protein, with an apparent molecular mass of 30 kDa (calculated mass, 29.5 kDa).

FIG. 5.

UV-visible absorbance spectra of reconstituted MMP0704-His₆ and MMP0704(20-289)-His₆ holoproteins. The MMP0704-His₆ apoprotein (broken line, bottom spectrum) was treated with ferric chloride, sulfide, and Mg-ATP and then desalted to reconstitute the holoprotein (solid line, top spectrum). The MMP0704(20-289)-His₆ apoprotein had an absorbance spectrum identical to that of the full-length apoprotein (data not shown), while the truncated holoprotein had an intermediate absorbance spectrum (dashed line, middle spectrum). The millimolar absorption coefficient (ɛ) was calculated from measured absorbance and protein concentration values.