Cupriavidus metallidurans is adapted to high concentrations of transition metal cations and is a model system for studying metal homeostasis in difficult environments. The elemental composition of C. metallidurans cells cultivated under various conditions was determined, revealing the ability of the bacterium to shield homeostasis of one essential metal from the toxic action of another. The contribution of metal uptake systems to this ability was studied. C. metallidurans contains three CorA members of the metal inorganic transport (MIT) protein family of putative magnesium uptake systems, ZupT of the ZRT/IRT protein, or ZIP, family, and PitA, which imports metal phosphate complexes. Expression of the genes for all these transporters was regulated by zinc availability, as shown by reporter gene fusions. While expression of zupT was upregulated under conditions of zinc starvation, expression of the other genes was downregulated at high zinc concentrations. Only corA 1 expression was influenced by magnesium starvation. Deletion mutants were constructed to characterize the contribution of each system to transition metal import. This identified ZupT as the main zinc uptake system under conditions of low zinc availability, CorA 1 as the main secondary magnesium uptake system, and CorA 2 and CorA 3 as backup systems for metal cation import. PitA may function as a cation-phosphate uptake system, the main supplier of divalent metal cations and phosphate in phosphate-rich environments. Thus, metal homeostasis in C. metallidurans is achieved by highly redundant metal uptake systems, which have only minimal cation selectivity and are in combination with efflux systems that "worry later" about surplus cations.Sophisticated cellular biochemistry needs metals as cofactors. About 40% of all enzymes have them, ranking from Mg (16%) Ͼ Zn (9%) Ͼ Fe (8%) Ͼ Mn (6%) Ͼ Ca (2%) Ͼ Co and Cu (1%) down to K, Na, Ni, V, Mo, W, and only one example of Cd (59). It is an interesting question how the correct metal is allocated to the right protein, a challenge especially for the divalent metal cations Mg 2ϩ , Zn 2ϩ , Fe 2ϩ , Mn 2ϩ , Co 2ϩ , Ni 2ϩ , and Cu 2ϩ . These metals compete with each other for the metal binding sites in enzymes (16). Additionally, Fe 2ϩ/3ϩ and Cu ϩ/2ϩ promote dangerous reactive oxygen species in Fenton and Fenton-like reactions, as described by Haber and Weiss (13).Part of the solution to this problem might be to keep the metal cation bouquet in any cellular compartment in a way that minimizes competition for metal binding sites and the Fenton reaction. This leads to the question of how the cellular metal cation bouquet can be maintained in environments that may contain a single metal in a concentration range from pM to mM. The betaproteobacterium Cupriavidus metallidurans strain CH34 is able to keep its metal homeostasis under a variety of such adverse conditions (19,28,30). The organism can be found in many mesophilic metal-contaminated environments around the globe, such as zinc deserts of Belgium (8). Key to this a...

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[NiFe]-hydrogenases bind a NiFe-(CN)2CO cofactor in their catalytic large subunit. The iron-sulfur protein HypD and the small accessory protein HypC play a central role in the generation of the CO and CN(-) ligands. Infrared spectroscopy identified signatures on an anaerobically isolated HypCD complex that are reminiscent of those in the hydrogenase active site, suggesting that this complex is the assembly site of the Fe-(CN)2CO moiety of the cofactor prior to its transfer to the hydrogenase large subunit. Here, we report that HypD isolated in the absence of HypC shows infrared bands at 1956 cm(-1), 2072 cm(-1), and 2092 cm(-1) that can be assigned to CO, CN(1), and CN(2), respectively, and which are indistinguishable from those observed for the HypCD complex. HypC could not be isolated with CO or CN(-) ligand contribution. Treatment of HypD with EDTA led to the concomitant loss of Fe and the CO and CN(-) signatures, while oxidation by H2O2 resulted in a positive shift of the CO and CN(-) bands by 35 cm(-1) and 20 cm(-1), respectively, indicative of the ferrous iron as an immediate ligation site for the diatomic ligands. Analysis of HypD amino acid variants identified cysteines 41, 69, and 72 to be essential for maturation of the cofactor. We propose a refined model for the ligation of Fe-(CN)2CO to HypD and the role of HypC in [NiFe]-hydrogenase maturation.

Cupriavidus metallidurans strain CH34 accomplishes a high level of transition metal resistance by a combination of rather unspecific transition metal import and controlled efflux of surplus metals. Using the plasmid-free mutant strain AE104 that possesses only a limited number of metal efflux systems, cellular metal pools were identified as counterparts of these transport reactions. At low zinc concentrations strain AE104 took up Zn(II) until the zinc content reached an optimum level of 70,000 Zn(II) per cell in the exponential phase of growth, whereas a ΔzupT mutant lacking the zinc importer ZupT contained only 20,000 Zn(II)/cell, possibly the minimum zinc content. Mutant and parent cells accumulated up to 125,000 Zn(II) per cell at high (100 μM) external zinc concentrations (optimum zinc content). When the mutant strain Δe4, which has all the known genes for zinc efflux systems deleted, was cultivated in the presence of zinc concentrations close to its upper tolerance level (10 μM), these cells contained 250,000 Zn(II) per cell, probably the maximum zinc content. Instead of zinc, 120,000 cobalt or cadmium ions could also fill-up parts of this zinc pool, showing that it is in fact an undefined pool of divalent transition metal cations bound with low substrate specificity. Even when the cells contained sufficient numbers of total zinc, the zinc importer ZupT was required for important cellular processes, indicating the presence of a pool of tightly bound zinc ions, which depends on ZupT for efficient replenishment. The absence of ZupT led to the formation of inclusion bodies, perturbed oxidative stress resistance and decreased efficiency in the synthesis of the zinc-dependent subunit RpoC of the RNA polymerase, leading to RpoC accumulation. Moreover, when a czc allele for a zinc-exporting transenvelope efflux system CzcCBA was constitutively expressed in a ΔzupT mutant, this led to the disappearance of the CzcA protein and the central subunit of the protein complex, and to the loss of metal resistance. This phenomenon occurred only if the ΔzupT cells had been cultivated for several transfers in solid culture medium, which generated severe zinc starvation. Thus, two zinc pools appear to exist in C. metallidurans: one pool of at least 20,000 tightly bound Zn(II) ions, in addition to a second pool of up to 200,000 cations bound loosely and with low substrate specificity.

a b s t r a c tThe HypC and HypD maturases are required for the biosynthesis of the Fe(CN) 2 CO cofactor in the large subunit of [NiFe]-hydrogenases. Using infrared spectroscopy we demonstrate that an anaerobically purified, Strep-tagged HypCD complex from Escherichia coli exhibits absorption bands characteristic of diatomic CO and CN À ligands as well as CO 2 . Metal and sulphide analyses revealed that along with the [4Fe-4S] 2+ cluster in HypD, the complex has two additional oxygen-labile Fe ions. We prove that HypD cysteine 41 is required for the coordination of all three ligands. These findings suggest that the HypCD complex carries minimally the Fe(CN) 2 CO cofactor.