Elsevier

Journal of Inorganic Biochemistry

Volume 115, October 2012, Pages 148-154
Journal of Inorganic Biochemistry

The X-ray crystal structure of a pseudoazurin from Sinorhizobium meliloti

https://doi.org/10.1016/j.jinorgbio.2012.04.005Get rights and content

Abstract

The X-ray crystal structure of oxidised pseudoazurin from the denitrifying plant symbiotic bacterium Sinorhizobium meliloti (SmPAz2) has been solved to a resolution of 2.0 Å. The pseudoazurin from Sinorhizobium sp. is unusual as it forms an operon with a sulfite dehydrogenase enzyme, rather than a Cu nitrite reductase. Examination of the structure reveals that the geometric parameters of the Type I Cu site in SmPAz2 correlate with observed features in the electronic spectrum of the protein. Comparison of the structure of SmPAz2 with those of pseudoazurins from five other bacterial species shows that the surface of SmPAz2 bears a conserved hydrophobic patch encircled by positively-charged residues, which may serve as a recognition site for its redox partners.

Graphical abstract

The crystal structure of a pseudoazurin from Sinorhizobium meliloti, shows a typical pseudoazurin fold and a conserved electrostatic surface structure, indicating the possible site of its interaction with its redox partners.

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Introduction

The determination of the first X-ray crystal structure of a blue copper protein (poplar plastocyanin [1]) was arguably the highlight of Hans Freeman's distinguished career in bioinorganic chemistry, and resulted from his pioneering work in establishing the first protein crystallography laboratory in Australia at the University of Sydney. In this paper, we continue in the spirit of Hans Freeman's research, with the determination of the structure of a blue copper protein—that of a pseudoazurin from Sinorhizobium meliloti to 2.0 Å resolution.

Pseudoazurin is a small (~ 15 kDa) blue copper protein found in a number of denitrifying and methylotrophic bacteria [2], [3]. The protein was first isolated from Achromobacter cycloclastes IAM1013 [4] and subsequently named pseudoazurin by Ambler and Tobari [5]. To date, structures of pseudoazurins from five species of bacteria have been solved: Alcaligenes faecalis [6], Achromobacter cycloclastes [7], Paracoccus pantotrophus [8], Methylobacterium extorquens [9] and Hyphomicrobium denitrificans [10]. The overall fold of these pseudoazurins has been found to incorporate an eight-stranded β‐barrel at the N-terminus of the protein, common to all blue copper proteins, and two C‐terminal α-helices, a feature unique to pseudoazurins [11]. The β-barrel structure incorporates four conserved residues that act as copper ligands to form the Type I copper coordination site: a cysteine, a methionine, and two histidine residues.

In all known pseudoazurin structures, the copper coordination site has a distorted tetrahedral coordination geometry. In their oxidised [Cu(II)] state, pseudoazurins are characterised by an intense optical absorption at ~ 600 nm, which is attributed to a charge-transfer transition from the thiolate sulfur of the coordinating cysteine residue to the Cu(II) atom [12] as well as additional peaks near 450 and 750 nm. In purified form pseudoazurins, such as that from H. denitrificans, which has a relatively high A450/A600 ratio, appear green in colour, rather than blue. Pseudoazurins are characterised by relatively high redox potentials (+ 200 to + 270 mV at pH 7.0, (Table 1 [13]) when compared with small cupric complexes; the value of which is influenced by the structures of both the inner and outer copper coordination spheres.

Pseudoazurin is produced in methylotrophs in the presence of high copper concentrations, when the cells are grown in the presence of methanol. In denitrifying bacteria, pseudoazurin functions as an electron donor to several enzymes of the denitrification pathway, including the copper nitrite reductase (CuNIR), nitrous oxide reductase and nitric oxide reductase, as well as other enzymes such as cytochrome c peroxidase [8]. In all pseudoazurin structures reported to date, the copper binding site is situated ~ 5 Å from the exterior of the protein in a location that facilitates efficient electron transfer to and from its redox partners. The adjacent molecular surface is characterised by a distinctive “hydrophobic patch”, encircled by a number of basic amino acid residues, most often lysine. In pseudoazurins from denitrifying bacteria, this region of the surface provides a recognition site for its CuNIR electron-transfer partner [3], [14]. Structural investigations have shown that following the formation of the encounter complex, via interactions between complementary surfaces on the two proteins, the redox centres of pseudoazurin and the CuNIR are positioned in close proximity, promoting fast electron transfer [15]. The close functional relationship between the pseudoazurin and CuNIR is also reflected in the fact that the genes encoding the CuNIR and the pseudoazurin are normally found together as part of the same operon.

The genome of the denitrifying plant symbiont bacterium Sinorhizobium meliloti Rm1021 includes two pseudoazurin genes, one canonical and one unusual (SmPAz1 and SmPAz2, respectively) [16]. Unlike the pseudoazurins characterised previously, the SmPAz2 gene (Smc04047) is found downstream of two genes encoding a cytochrome c (Smc04048) and a sulfite dehydrogenase (Smc04049, SorT), rather than in proximity to genes encoding a CuNIR. The Smc04049 to Smc04047 genes form an operon and are co-transcribed [17].

A primary sequence comparison between SmPAz2 and the five pseudoazurins for which structures have been determined reveals that SmPAz2 possesses high sequence similarity to the other pseudoazurins (39–52%, Table 1, Fig. 1). The determined value for its redox potential at + 200 mV (pH 7.0) is the lowest among the structurally characterised proteins [18]. However, although this redox potential is in the same range as that determined for the Smc04048 cytochrome, investigations into possible interactions between the three proteins in the sulfite dehydrogenase operon revealed no electron transfer between purified SmPAz2 and SorT, while electron transfer between the Smc04048 cytochrome and SorT could be demonstrated [18]. In order to better understand the function of SmPAz2, we have solved and refined the crystal structure of this protein in the oxidised, Cu(II) form to 2.0 Å resolution.

Section snippets

Phylogenetic analyses

Protein sequences related to SmPAz2 were identified using BLAST [19] searches of publicly available databases. The sequences were aligned and phylogenetic trees constructed in MEGA 5.0 [20]. Robustness testing used the bootstrap method with 500 resampling cycles. The results are presented in Figure S1.

Protein overexpression and purification

Escherichia coli BL21(DE3) cells were transformed with the pETazu2T plasmid for pseudoazurin overexpression, as described previously [18] and expression cultures grown on LB medium supplemented

Overall structure of SmPAz2

SmPAz2 crystallised in space group P212121 with a single molecule in the asymmetric unit. Refinement of the model to 2.0 Å resolution converged with residuals R = 0.202 and Rfree = 0.261, and excellent statistics and model geometry (Table 2). The atomic coordinates and structure factors have been deposited into the Protein Data Bank (PDB) with the accession code 3TU6. While the mature, native pseudoazurin consists of residues 22–147 following cleavage of the N-terminal periplasmic localisation

Conclusion

The structure of SmPAz2 has been solved by X-ray crystallography to 2.0 Å resolution. The solution of the structure enabled detailed comparisons with five structurally characterised pseudoazurins, for which functions and spectroscopic characteristics have been described. This analysis showed that the distorted tetrahedral geometry of the Cu coordination site in SmPAz2, with shorter Met Sδ―Cu and longer Cys Sγ―Cu bonds than the corresponding distances in other pseudoazurins is reflected in

Abbreviations

    SmPAz1

    Sinorhizobium meliloti pseudoazurin 1

    SmPAz2

    Sinorhizobium meliloti pseudoazurin 2

    CuNIR

    copper-containing nitrite reductase

    Tris

    2-Amino-2-hydroxymethyl-propane-1,3-diol

    BCA

    bicinchoninic acid

    SorT

    sulfite dehydrogenase from Sinorhizobium meliloti

    PEG MME

    polyethylene glycol monomethyl ether

    PDB

    Protein Data Bank

Acknowledgements

We thank Dr Miriam-Rose Ash for her careful reading of the manuscript. This study was supported by the ARC fellowship and grant (DP0878525) to UK. This research was undertaken on the MX1 and MX2 beamlines at the Australian Synchrotron, Victoria, Australia. M.J.M. is supported by a LIMS Senior Research Fellowship.

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    These authors contributed equally to this work.

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