Zn, Cu and Co in cyanobacteria: selective control of metal availability.

Homeostatic systems for essential and non-essential metals create the cellular environments in which the correct metals are acquired by metalloproteins while the incorrect ones are somehow avoided. Cyanobacteria have metal requirements often absent from other bacteria; copper in thylakoidal plastocyanin, zinc in carboxysomal carbonic anhydrase, cobalt in cobalamin but magnesium in chlorophyll, molybdenum in heterocystous nitrogenase, manganese in thylakoidal water-splitting oxygen-evolving complex. This article reviews: an intracellular trafficking pathway for inward copper supply, the sequestration of surplus zinc by metallothionein (also present in other bacteria) and the detection and export of excess cobalt. We consider the influence of homeostatic proteins on selective metal availability.

A novel copper site in a cyanobacterial metallochaperone.

The thylakoid lumen of the cyanobacterium Synechocystis PCC 6803 is supplied with copper via two copper-transporting ATPases and a metallochaperone intermediary. We show that the copper site of this metallochaperone is unusual and consists of two cysteine residues and a histidine imidazole located on structurally dynamic loops. Substitution of this histidine residue enhances bacterial two-hybrid interaction with the cytosolic copper exporter, but not the copper importer, suggesting that the interacting surfaces are distinct, with implications for metal transfer.

CopZ from Bacillus subtilis interacts in vivo with a copper exporting CPx-type ATPase CopA.

The structure of the hypothetical copper-metallochaperone CopZ from Bacillus subtilis and its predicted partner CopA have been studied but their respective contributions to copper export, -import, -sequestration and -supply are unknown. DeltacopA was hypersensitive to copper and contained more copper atoms cell(-1) than wild-type. Expression from the copA operator-promoter increased in elevated copper (not other metals), consistent with a role in copper export. A bacterial two-hybrid assay revealed in vivo interaction between CopZ and the N-terminal domain of CopA but not that of a related transporter, YvgW, involved in cadmium-resistance. Activity of copper-requiring cytochrome caa(3) oxidase was retained in deltacopZ and deltacopA. DeltacopZ was only slightly copper-hypersensitive but deltacopZ/deltacopA was more sensitive than deltacopA, implying some action of CopZ that is independent of CopA. Significantly, deltacopZ contained fewer copper atoms cell(-1) than wild-type under these conditions. CopZ makes a net contribution to copper sequestration and/or recycling exceeding any donation to CopA for export.

Chimeras of P-type ATPases and their transcriptional regulators: contributions of a cytosolic amino-terminal domain to metal specificity.

Zn(2+)-responsive repressor ZiaR and Co(2+)-responsive activator CoaR modulate production of P(1)-type Zn(2+)- (ZiaA) and Co(2+)- (CoaT) ATPases respectively. What dictates metal selectivity? We show that Delta ziaDeltacoa double mutants had similar Zn(2+) resistance to Deltazia single mutants and similar Co(2+) resistance to Deltacoa single mutants. Controlling either ziaA or coaT with opposing regulators restored no resistance to metals sensed by the regulators, but coincident replacement of the deduced cytosolic amino-terminal domain CoaT(N) with ZiaA(N) (in ziaR-(p) ziaA-ziaA(N)coaT) conferred Zn(2+) resistance to DeltaziaDeltacoa, Zn(2+) content was lowered and residual Co(2+) resistance lost. Metal-dependent molar absorptivity under anaerobic conditions revealed that purified ZiaA(N) binds Co(2+) in a pseudotetrahedral two-thiol site, and Co(2+) was displaced by Zn(2+). Thus, the amino-terminal domain of ZiaA inverts the metals exported by zinc-regulated CoaT from Co(2+) to Zn(2+), and this correlates simplistically with metal-binding preferences; K(ZiaAN) Zn(2+) tighter than Co(2+). However, Zn(2+) did not bleach Cu(+)-ZiaA(N), and only Cu(+) co-migrated with ZiaA(N) after competitive binding versus Zn(2+). Bacterial two-hybrid assays that detected interaction between the Cu(+)-metallochaperone Atx1 and the amino-terminal domain of Cu(+)-transporter PacS(N) detected no interaction with the analogous, deduced, ferredoxin-fold subdomain of ZiaA(N). Provided that there is no freely exchangeable cytosolic Cu(+), restricted contact with the Cu(+)-metallochaperone can impose a barrier impairing the formation of otherwise favoured Cu(+)-ZiaA(N) complexes.

Solution structures of a cyanobacterial metallochaperone: insight into an atypical copper-binding motif.

The Atx1 copper metallochaperone from Synechocystis PCC 6803, ScAtx1, interacts with two P(1)-type copper ATPases to supply copper proteins within intracellular compartments, avoiding ATPases for other metals en route. Here we report NMR-derived solution structures for ScAtx1. The monomeric apo form has a betaalphabetabetaalpha fold with backbone motions largely restricted to loop 1 containing Cys-12 and Cys-15. The tumbling rate of Cu(I)ScAtx1 (0.1-0.8 mm) implies dimers. Experimental restraints are satisfied by symmetrical dimers with Cys-12 or His-61, but not Cys-15, invading the copper site of the opposing subunit. A full sequence of copper ligands from the cell surface to thylakoid compartments is proposed, considering in vitro homodimer liganding to mimic in vivo liganding in ScAtx1-ATPase heterodimers. A monomeric high resolution structure for Cu(I)ScAtx1, with Cys-12, Cys-15, and His-61 as ligands, is calculated without violations despite the rotational correlation time. (2)J(NH) couplings in the imidazole ring of His-61 establish coordination of N(epsilon2) to copper. His-61 is analogous to Lys-65 in eukaryotic metallochaperones, stabilizing Cu(I)S(2) complexes but by binding Cu(I) rather than compensating charge. Cys-Cys-His ligand sets are an emergent theme in some copper metallochaperones, although not in related Atx1, CopZ, or Hah1. Surface charge (Glu-13) close to the metal-binding site of ScAtx1 is likely to support interaction with complementary surfaces of copper-transporting ATPases (PacS-Arg-11 and CtaA-Lys-14) but to discourage interaction with zinc ATPase ZiaA and so inhibit aberrant formation of copper-ZiaA complexes.

Surplus zinc is handled by Zym1 metallothionein and Zhf endoplasmic reticulum transporter in Schizosaccharomyces pombe.

Homeostatic mechanisms prevent the accumulation of free zinc in the cytoplasm, raising questions regarding where surplus zinc is stored and how it is delivered to and from these stores. A genetic screen for zinc hypersensitivity in Schizosaccharomyces pombe identified a missense mutation truncating Zhf, an endoplasmic reticulum transporter. These cells were approximately 5-fold more zinc-sensitive than other independent mutants. The targeted disruption of zhf prevented growth on low zinc medium and caused hypersensitivity to elevated zinc/cobalt but resistance to cadmium. The exposure to elevated zinc but not copper also promotes the accumulation of transcripts encoding a metallothionein designated Zym1. The Sty1 pathway is required for maximal zym1 expression but is not obligatory for zinc perception. The targeted disruption of zym1 impaired cadmium tolerance but only slightly impaired zinc tolerance, whereas zym1 overexpression substantially rescued zinc hypersensitivity of zhf(-) cells. Four equivalents of zinc were displaced from Zym1 by up to 12 equivalents of p-(hydroxymercuri)phenylsulphonate. Zym1 thiols react rapidly with 5,5'-dithiobis-(2-nitrobenzoic acid) compared with bacterial zinc metallothionein (6.8 and 0.2 x 10(-4) s(-1), respectively). Zym1 is unlike known fungal metallothioneins that are induced by and sequester copper but not zinc. Less zinc but normal cadmium was accumulated by zym1Delta, consistent with zinc sequestration by Zym1 in vivo.

A copper metallochaperone for photosynthesis and respiration reveals metal-specific targets, interaction with an importer, and alternative sites for copper acquisition.

A bacterial two-hybrid assay revealed interaction between a protein now designated bacterial Atx1 and amino-terminal domains of copper-transporting ATPases CtaA (cellular import) and PacS (thylakoid import) but not the related zinc (ZiaA) or cobalt (CoaT) transporters from the same organism (Synechocystis PCC 6803). The specificity of metallochaperone interactions coincides with metal specificity. After reconstitution in a N(2) atmosphere, bacterial Atx1 bound 1 mol of copper mol(-1), and apoPacS(N) acquired copper from copper-Atx1. Copper was displaced from Atx1 by p-(hydroxymercuri)phenylsulfonate, indicative of thiol ligands, and two cysteine residues were obligatory for two-hybrid interaction with PacS(N). This organism contains compartments (thylakoids) where the copper proteins plastocyanin and cytochrome oxidase reside. In copper super-supplemented mutants, photooxidation of cytochrome c(6) was greater in Deltaatx1DeltactaA than in DeltactaA, showing that Atx1 contributes to efficient switching from iron in cytochrome c(6) to copper in plastocyanin for photosynthetic electron transport. Cytochrome oxidase activity was also less in membranes purified from low [copper]-grown Deltaatx1 or DeltapacS, compared with wild-type, but the double mutant Deltaatx1DeltapacS was non-additive, consistent with Atx1 acting via PacS. Conversely, activity in Deltaatx1DeltactaA was less than in either respective single mutant, revealing that Atx1 can function without the major copper importer and consistent with a role in recycling endogenous copper.

Characterization of the cobaltochelatase CbiXL: evidence for a 4Fe-4S center housed within an MXCXXC motif.

CbiX is a cobaltochelatase required for the biosynthesis of vitamin B12 and is found in Archaea as a short form (CbiXS containing 120-145 amino acids) and in some bacteria as a longer version (CbiXL containing 300-350 amino acids). Purification of either recombinant Bacillus megaterium or Synechocystis CbiXL in Escherichia coli, which is facilitated by the presence of a naturally occurring histidine-rich region of the protein, results in the isolation of a dark brown protein solution. The UV/visible spectrum of the protein is consistent with the presence of a redox group, and the lack of definition within the spectrum is suggestive of a 4Fe-4S center. The presence of an iron-sulfur center was confirmed by EPR analysis of the proteins, which produces a pseudoaxial spectrum with g values at 2.04, 1.94, and 1.90. The EPR spectrum was absent at 70 K, an observation that is diagnostic of a 4Fe-4S center. Redox potentiometry coupled with optical spectroscopy allowed the midpoint potential of the redox center to be determined for the CbiXL from both B. megaterium and Synechocystis. Sequence analysis of CbiXL proteins reveals only two conserved cysteine residues within the CbiXL proteins, which are part of an MXCXXC motif. Mutagenesis of the two cysteines leads to loss of both the EPR spectrum and UV/visible spectral features of the Fe-S center in the protein, clearly indicating that these residues are involved in ligating the cofactor to the apoprotein possibly in a butterfly arrangement. The potential physiological role of the iron-sulfur center is discussed.