Arabidopsis thaliana GLX2-1 contains a dinuclear metal binding site, but is not a glyoxalase 2

Abstract

In an effort to probe the structure and function of a predicted mitochondrial glyoxalase 2, GLX2-1, from Arabidopsis thaliana, GLX2-1 was cloned, overexpressed, purified and characterized using metal analyses, kinetics, and UV-visible, EPR, and (1)H-NMR spectroscopies. The purified enzyme was purple and contained substoichiometric amounts of iron and zinc; however, metal-binding studies reveal that GLX2-1 can bind nearly two equivalents of either iron or zinc and that the most stable analogue of GLX2-1 is the iron-containing form. UV-visible spectra of the purified enzyme suggest the presence of Fe(II) in the protein, but the Fe(II) can be oxidized over time or by the addition of metal ions to the protein. EPR spectra revealed the presence of an anti-ferromagnetically-coupled Fe(III)Fe(II) centre and the presence of a protein-bound high-spin Fe(III) centre, perhaps as part of a FeZn centre. No paramagnetically shifted peaks were observed in (1)H-NMR spectra of the GLX2-1 analogues, suggesting low amounts of the paramagnetic, anti-ferromagnetically coupled centre. Steady-state kinetic studies with several thiolester substrates indicate that GLX2-1 is not a GLX2. In contrast with all of the other GLX2 proteins characterized, GLX2-1 contains an arginine in place of one of the metal-binding histidine residues at position 246. In order to evaluate further whether Arg(246) binds metal, the R246L mutant was prepared. The metal binding results are very similar to those of native GLX2-1, suggesting that a different amino acid is recruited as a metal-binding ligand. These results demonstrate that Arabidopsis GLX2-1 is a novel member of the metallo-beta-lactamase superfamily.

Figure 1

Structure of mitochondrial GLX2−5

Figure 2. Alignment of predicted plant GLXII proteins

The metallo-β-lactamase fold motif is shaded in grey, and substrate-binding ligands are indicated by triangles. Metal-binding ligands are indicated by #, * indicates the β-lactamase fold motif.

Figure 3

UV–visible spectrum of 1.2 mM as-isolated GLX2−1 in 10 mM Mops, pH 7.2

Figure 4. EPR spectra of GLX2−1

Trace A is the EPR spectrum of as-isolated GLX2−1. Trace B is a computer simulation assuming two spin-coupled iron ions in an Fe(III)Fe(II) centre. Trace C is the spectrum of GLX2−1 after aerobic addition of Fe(II). Trace D is an expanded view of Trace A and Trace E is of GLX2−1 after aerobic addition of Fe(II). Experimental conditions were: A, 12 K, 2 mW; C, 10 K, 1 mW; D, 12 K, 2 mW; E, 10 K, 20 mW. Spectra A and C are shown normalized for temperature and microwave power; spectra D and E are shown with arbitrary intensities. 10 G (1 mT) magnetic field modulation at 100 kHz was employed and other parameters were chosen such that spectral resolution was limited by the field modulation. The parameters used for the simulation of Trace B were: for Fe(III), $S=\frac{5}{2}$, g_isotropic = 2.0, D = 2 cm⁻¹ and E/D = 0; for Fe(II), S = 2, g_(x,y,z) = 1.970, 2.090, 2.025, D = 15 cm⁻¹ and E/D = 0.2; for the dinuclear centre, J = 43 cm⁻¹ and an inter-iron distance, r, of 3.6 Å (0.36 nm) was assumed. It should be noted that (i) this is not a unique solution, and (ii) the simulation was not sensitive to all the parameters; in particular, the dependence on r and on which iron was assumed to have a rhombic zero-field splitting term was slight.