Characterization of functional heme domains from soluble guanylate cyclase

Abstract

Soluble guanylate cyclase (sGC) is a heterodimeric, nitric oxide (NO)-sensing hemoprotein composed of two subunits, alpha1 and beta1. NO binds to the heme cofactor in the beta1 subunit, forming a five-coordinate NO complex that activates the enzyme several hundred-fold. In this paper, the heme domain has been localized to the N-terminal 194 residues of the beta1 subunit. This fragment represents the smallest construct of the beta1 subunit that retains the ligand-binding characteristics of the native enzyme, namely, tight affinity for NO and no observable binding of O(2). A functional heme domain from the rat beta2 subunit has been localized to the first 217 amino acids beta2(1-217). These proteins are approximately 40% identical to the rat beta1 heme domain and form five-coordinate, low-spin NO complexes and six-coordinate, low-spin CO complexes. Similar to sGC, these constructs have a weak Fe-His stretch [208 and 207 cm(-)(1) for beta1(1-194) and beta2(1-217), respectively]. beta2(1-217) forms a CO complex that is very similar to sGC and has a high nu(CO) stretching frequency at 1994 cm(-)(1). The autoxidation rate of beta1(1-194) was 0.073/min, while the beta2(1-217) was substantially more stable in the ferrous form with an autoxidation rate of 0.003/min at 37 degrees C. This paper has identified and characterized the minimum functional ligand-binding heme domain derived from sGC, providing key details toward a comprehensive characterization.

Figure 1

Alignment of rat β1(1-194) with rat β2(1-217) and the Thermoanaerobacter tengcogensis H-NOX domain. The alignment was generated using DNASTAR's Megalign program and the Clustal W algorithm. Conserved residues that are identical to the consensus sequence are shaded in black. Conserved residues discussed in the text are marked with an asterisk (His105, the heme ligand, and Tyr135, Ser137 and Arg139 using the rat β1 numbering).

Figure 2

Electronic absorption spectra of β1(1-194) and β2(1-217) showing the Soret maximum and α/β region. Reduced, unligated (—); CO complex (- - -); and NO complex (^………). Heme concentration was ∼7 μM.

Figure 3

β1(1-194) resonance Raman spectra. Fe⁺²-unligated in the low (a) and high frequency region (h); ¹⁴NO and ¹⁵NO complexes and their difference spectra in the low (b, c and d) and high frequency regions (i, j and k), respectively; ¹²CO and ¹³CO complexes and their difference spectra in the low (e, f and g) and high frequency regions (l and m), respectively. Heme concentration was 14 μM. The Raman intensity was normalized to the ν₄ mode. The asterisks indicate subtraction artifacts.

Figure 4

β2(1-217) resonance Raman spectra. Fe⁺²-unligated in the low (a) and high frequency region (h); ¹⁴NO and ¹⁵NO complexes and their difference spectra in the low (b, c and d) and high frequency regions (i, j and k), respectively; ¹²CO and ¹³CO complexes and their difference spectra in the low (e, f and g) and high frequency regions (l and m), respectively. Heme concentration was 19 μM. The Raman intensity was normalized to the ν₄ mode. The asterisks indicate subtraction artifacts.

Figure 5

Autoxidation rates of β1(1-194) and β2(1-217). Shown in panel A are difference spectra associated with the autoxidation of β1(1-194) and panel B shows a plot of the Δ434 – Δ404 versus time and a fit (grey line) to the data from which a rate constant was derived. Panel C shows the difference spectra for the autoxidation of β2(1-217) and panel D shows a plot of Δ432 - Δ390 versus time and a fit to those data (grey line).

Figure 6

Homology models of the distal pockets. Homology models of the β1 and β2 were generated using MODELLER following the methods described in (13). The view in the model is from below the heme and looking up into the distal pocket. H-bonding donors or residues that would provide negative polarity are shown in light grey. Tyr140 in the TtTar4H distal site is clearly visible, however, there is none visible in either model of β1(1-194) or β2(1-217).

Figure 7

ν(Fe-CO)/ν(CO) correlation for 6-coordinate CO adducts of model porphyrins and hemoproteins. Values for the metalloporphyrins and hemoproteins were obtained from: Kerr et al., Table 2, complexes 1-15 (38); Table 3 in this work; CooA (39, 40); EcDosH (24); BjFixL (24); Heme oxygenase (41, 42); Sperm whale (Sw) Mb (21); Mb (H64V/V68T) (37). For sGC, β1(1-385) and β1(1-194), the numeric suffix refers to the CO conformation with the low and high ν(Fe-CO) frequency (1 and 2, respectively).