Structure and properties of a bis-histidyl ligated globin from Caenorhabditis elegans

Abstract

Globins are heme-containing proteins that are best known for their roles in oxygen (O(2)) transport and storage. However, more diverse roles of globins in biology are being revealed, including gas and redox sensing. In the nematode Caenorhabditis elegans, 33 globin or globin-like genes were recently identified, some of which are known to be expressed in the sensory neurons of the worm and linked to O(2) sensing behavior. Here, we describe GLB-6, a novel globin-like protein expressed in the neurons of C. elegans. Recombinantly expressed full-length GLB-6 contains a heme site with spectral features that are similar to those of other bis-histidyl ligated globins, such as neuroglobin and cytoglobin. In contrast to these globins, however, ligands such as CO, NO, and CN(-) do not bind to the heme in GLB-6, demonstrating that the endogenous histidine ligands are likely very tightly coordinated. Additionally, GLB-6 exhibits rapid two-state autoxidation kinetics in the presence of physiological O(2) levels as well as a low redox potential (-193 +/- 2 mV). A high-resolution (1.40 A) crystal structure of the ferric form of the heme domain of GLB-6 confirms both the putative globin fold and bis-histidyl ligation and also demonstrates key structural features that can be correlated with the unusual ligand binding and redox properties exhibited by the full-length protein. Taken together, the biochemical properties of GLB-6 suggest that this neural protein would most likely serve as a physiological sensor for O(2) in C. elegans via redox signaling and/or electron transfer.

Figure 1

UV-Vis absorption spectra of full-length ferrous GLB-6 (solid line) and ferric GLB-6 (dotted line). (Buffer = 20 mM HEPES, 150 mM NaCl, pH 7.5)

Figure 2

Resonance Raman spectra of full-length ferrous GLB-6 (a, c) and ferric GLB-6 (b, d). The left panel (traces a and b) shows the lower frequency region and is normalized to ν₇. The right panel (traces c and d) shows the high frequency region and is normalized to ν₄. All spectra were acquired for 1 hour at room temperature with 413.1 nm excitation, room temperature, 2 mW.

Figure 3

Autoxidation rates of full-length GLB-6 measured by stopped-flow spectroscopy. (a) Spectral changes with time for the oxidation of ferrous GLB-6. Experiments were performed at 10 °C with different O₂ concentrations, 5.25, 10.5, 21.0, 37.1, and 50.0 % O₂. The spectral changes at 5.25 % O₂ are shown as representative data. The curve fitting of the change in A₄₂₅ versus time using a single exponential model is shown in the inset. (b) The observed first-order autoxidation rate constant k_obs of full-length GLB-6 is plotted as a function of O₂ concentration. The fit to a linear function is shown as a dotted line.

Figure 4

Spectroelectrochemical potentiometric redox titrations of full-length GLB-6. The intensity of the α-band maxima, A₅₆₀, of ferrous GLB-6 minus A₅₆₀ of ferric GLB-6 is normalized and plotted versus the ambient potential (inset). The data were analyzed for the best fit to Nernst equations with n = 1 (inset, dotted line). The measured redox potential versus SHE, E⁰’, is −193 ± 2 mV, which is an average of three experiments. (Buffer: 100 mM potassium phosphate, 150 mM NaCl, pH 7.0, Temperature = 25 °C).

Figure 5

Structure of the heme domain of GLB-6. Ribbon structure of the GLB-6 heme domain (molecule A) is shown with helices labeled according to the canonical globin fold. His254 and His289, distal (above) and proximal (below) heme ligands, respectively, and the heme moiety are shown as stick representation. Praseodymium atoms are represented as orange spheres. Note that residues 318–321 could not be traced and are shown as black dots. Lower panel shows 2F_o-F_c composite simulated-annealing omit electron density map (1.0σ, gray mesh) calculated with heme moieties omitted.

Figure 6

Heme (a) and overall (b) alignments of GLB-6 (red) with murine neuroglobin (yellow, PDB: 1Q1F) (top) and Geobacter sulfurreducens globin-coupled sensor (blue, PDB: 2W31) (bottom). Only residue assignments for GLB-6 are provided for clarity. Helices are labeled according to Figure 5. D-and Z-helices are marked with an asterisk to indicate that they do not exist in the GLB-6 structure.