The effects of nitroxyl (HNO) on soluble guanylate cyclase activity: interactions at ferrous heme and cysteine thiols

It has been previously proposed that nitric oxide (NO) is the only biologically relevant nitrogen oxide capable of activating the enzyme soluble guanylate cyclase (sGC). However, recent reports implicate HNO as another possible activator of sGC. Herein, we examine the affect of HNO donors on the activity of purified bovine lung sGC and find that, indeed, HNO is capable of activating this enzyme. Like NO, HNO activation appears to occur via interaction with the regulatory ferrous heme on sGC. Somewhat unexpectedly, HNO does not activate the ferric form of the enzyme. Finally, HNO-mediated cysteine thiol modification appears to also affect enzyme activity leading to inhibition. Thus, sGC activity can be regulated by HNO via interactions at both the regulatory heme and cysteine thiols

Mutation of Trimethyllysine 72 to Alanine Enhances His79–Heme-Mediated Dynamics of Iso-1-cytochrome <i>c</i>

Trimethyllysine 72 (Tml72) of yeast iso-1-cytochrome <i>c</i> lies across the surface of the heme crevice loop (Ω-loop D, residues 70–85) like a brace. Lys72 is oriented similarly in horse cytochrome <i>c</i> (Cyt<i>c</i>). To determine whether this residue affects the dynamics of opening the heme crevice loop, we have studied the effect of a Tml72 to Ala substitution on the formation of the His79–heme alkaline conformer near neutral pH using a variant of iso-1-Cyt<i>c</i> including K72A and K79H mutations. Guanidine hydrochloride denaturation shows that the Tml72 to Ala substitution within error does not affect the global stability of the protein. The effect of the Tml72 to Ala substitution on the thermodynamics of the His79–heme alkaline transition is also small. However, pH-jump kinetic studies of the His79–heme alkaline transition show that both the forward and backward rates of conformational change are increased by the Tml72 to Ala substitution. The barrier for opening the heme crevice is reduced by 0.5 kcal/mol and for closing the heme crevice by 0.3 kcal/mol. The ability of Tml72 to modulate the heme crevice dynamics may indicate a crucial role in regulating function, such as in the peroxidase activity seen in the early stages of apoptosis

Ferrous human cystathionine b-synthase loses activity during enzyme assay due to a ligand switch process

Cystathionine β-synthase (CBS) is a pyridoxal-5‘-phosphate-dependent enzyme that catalyzes the condensation of serine and homocysteine to form cystathionine. Mammalian CBS also contains a heme cofactor that has been proposed to allosterically regulate enzyme activity via the heme redox state, with FeII CBS displaying approximately half the activity of FeIII CBS in vitro. The results of this study show that human FeII CBS spontaneously loses enzyme activity over the course of a 20 min enzyme assay. Both the full-length 63-kDa and truncated 45-kDa form of CBS slowly and irreversibly lose activity upon reduction to the FeII form. Additionally, electronic absorption spectroscopy reveals that FeII CBS undergoes a heme ligand exchange to FeII CBS424 when the enzyme is incubated at 37 °C and pH 8.6. The addition of enzyme substrates or imidazole has a moderate effect on the rate of the ligand switch, but does not prevent conversion to the inactive species. Time-dependent spectroscopic data describing the conversion of FeII CBS to FeII CBS424 were fitted to a three-state kinetic model. The resultant rate constants were used to fit assay data and to estimate the activity of FeII CBS prior to the ligand switch. Based on this fit it appears that FeII CBS initially has the same enzyme activity as FeIII CBS, but FeII CBS loses activity as the ligand switch proceeds. The slow and irreversible loss of FeII CBS enzyme activity in vitro resembles protein denaturation, and suggests that a simple regulatory mechanism based on the heme redox state is unlikely

Probing Denatured State Conformational Bias in a Three-Helix Bundle, UBA(2), Using a Cytochrome <i>c</i> Fusion Protein

Previous work with the four-helix-bundle protein cytochrome <i>c</i>′ from Rhodopseudomonas palustris using histidine–heme loop formation methods revealed fold-specific deviations from random coil behavior in its denatured state ensemble. To examine the generality of this finding, we extend this work to a three-helix-bundle polypeptide, the second ubiquitin-associated domain, UBA(2), of the human DNA excision repair protein. We use yeast iso-1-cytochrome <i>c</i> as a scaffold, fusing the UBA(2) domain at the N-terminus of iso-1-cytochrome <i>c</i>. We have engineered histidine into highly solvent accessible positions of UBA(2), creating six single histidine variants. Guanidine hydrochloride denaturation studies show that the UBA(2)–cytochrome <i>c</i> fusion protein unfolds in a three-state process with iso-1-cytochrome <i>c</i> unfolding first. Furthermore, engineered histidine residues in UBA(2) strongly destabilize the iso-1-cytochrome <i>c</i> domain. Equilibrium and kinetic histidine–heme loop formation measurements in the denatured state at 4 and 6 M guanidine hydrochloride show that loop stability decreases as the size of the histidine–heme loop increases, in accord with the Jacobson–Stockmayer equation. However, we observe that the His27–heme loop is both more stable than expected from the Jacobson–Stockmayer relationship and breaks more slowly than expected. These results show that the sequence near His27, which is in the reverse turn between helices 2 and 3 of UBA­(2), is prone to persistent interactions in the denatured state. Therefore, consistent with our results for cytochrome <i>c</i>′, this reverse turn sequence may help to establish the topology of this fold by biasing the conformational distribution of the denatured state

Dynamics of the His79-Heme Alkaline Transition of Yeast Iso-1-cytochrome <i>c</i> Probed by Conformationally Gated Electron Transfer with Co(II)bis(terpyridine)

Alkaline conformers of cytochrome <i>c</i> may be involved in both its electron transport and apoptotic functions. We use cobalt­(II)­bis­(terpyridine), Co­(terpy)₂²⁺, as a reagent for conformationally gated electron-transfer (gated ET) experiments to study the alkaline conformational transition of K79H variants of yeast iso-1-cytochrome <i>c</i> expressed in Escherichia coli, WT*K79H, with alanine at position 72 and Saccharomyces cerevisiae, yK79H, with trimethyllysine (Tml) at position 72. Co­(terpy)₂²⁺ is well-suited to the 100 ms to 1 s time scale of the His79-mediated alkaline conformational transition of these variants. Reduction of the His79-heme alkaline conformer by Co­(terpy)₂²⁺ occurs primarily by gated ET, which involves conversion to the native state followed by reduction, with a small fraction of the His79-heme alkaline conformer directly reduced by Co­(terpy)₂²⁺. The gated ET experiments show that the mechanism of formation of the His79-heme alkaline conformer involves only two ionizable groups. In previous work, we showed that the mechanism of the His73-mediated alkaline conformational transition requires three ionizable groups. Thus, the mechanism of heme crevice opening depends upon the position of the ligand mediating the process. The microscopic rate constants provided by gated ET studies show that mutation of Tml72 (yK79H variant) in the heme crevice loop to Ala72 (WT*K79H variant) affects the dynamics of heme crevice opening through a small destabilization of both the native conformer and the transition state relative to the His79-heme alkaline conformer. Previous pH jump data had indicated that the Tml72→Ala mutation primarily stabilized the transition state for the His79-mediated alkaline conformational transition

The heme of cystathionine b-synthase likely undergoes a temperature-induced redox-mediated ligand switch

Cystathionine β-synthase (CBS) is a pyridoxal-5‘-dependent enzyme that catalyzes the condensation of homocysteine and serine to form cystathionine. Human CBS is unique in that heme is also required for maximal activity, although the function of heme in this enzyme is presently unclear. The study presented herein reveals that the heme of human CBS undergoes a coordination change upon reduction at elevated temperatures. We have termed this new species “CBS424” and demonstrate that its formation is likely irreversible when pH 9 FeIII CBS is reduced at moderately elevated temperatures (40 °C and higher) or when pH 9 FeII CBS is heated to similar temperatures. Spectroscopic techniques, including resonance Raman, electronic absorption, and variable temperature/variable field magnetic circular dichroism spectroscopy, provide strong evidence that CBS424 is coordinated by two neutral donor ligands. It appears likely that the native cysteine(thiolate) heme ligand is displaced by an endogenous neutral donor upon conversion to CBS424. This behavior is consistent with other six-coordinate, cysteine(thiolate)-ligated heme centers, which seek to avoid this coordination structure in the FeII state. Functional assays show that CBS424 is inactive and suggest that the ligand switch is responsible for eliminating enzyme activity. When this investigation is taken together with other functional studies of CBS, it provides strong evidence that coordination of Cys52 to the heme iron is crucial for full activity in this enzyme. We hypothesize that cysteine displacement may serve as a mechanism for CBS inactivation and that second-sphere interactions of the Cys52 thiolate with surrounding residues are responsible for communicating the heme ligand displacement to the CBS active site