Enzymes in the sulfur network generate the signaling molecule, hydrogen sulfide (H2S), from the amino acids cysteine and homocysteine. Since it is toxic at elevated concentrations, cells are equipped to clear H2S. A canonical sulfide oxidation pathway operates in mitochondria, converting H2S to thiosulfate and sulfate. We have recently discovered the ability of ferric hemoglobin to oxidize sulfide to thiosulfate and iron-bound hydropolysulfides. In this study, we report that myoglobin exhibits a similar capacity for sulfide oxidation. We have trapped and characterized iron-bound sulfur intermediates using cryo-mass spectrometry and X-ray absorption spectroscopy. Further support for the postulated intermediates in the chemically challenging conversion of H2S to thiosulfate and iron-bound catenated sulfur products is provided by EPR and resonance Raman spectroscopy in addition to density functional theory computational results. We speculate that the unusual sensitivity of skeletal muscle cytochrome c oxidase to sulfide poisoning in ethylmalonic encephalopathy, resulting from the deficiency in a mitochondrial sulfide oxidation enzyme, might be due to the concentration of H2S by myoglobin in this tissue.

Edited by Norma AllewellHydrogen sulfide is a critical signaling molecule, but high concentrations cause cellular toxicity. A four-enzyme pathway in the mitochondrion detoxifies H 2 S by converting it to thiosulfate and sulfate. Recent studies have shown that globins like hemoglobin and myoglobin can also oxidize H 2 S to thiosulfate and hydropolysulfides. Neuroglobin, a globin enriched in the brain, was reported to bind H 2 S tightly and was postulated to play a role in modulating neuronal sensitivity to H 2 S in conditions such as stroke. However, the H 2 S reactivity of the coordinately saturated heme in neuroglobin is expected a priori to be substantially lower than that of the 5-coordinate hemes present in myoglobin and hemoglobin. To resolve this discrepancy, we explored the role of the distal histidine residue in muting the reactivity of human neuroglobin toward H 2 S. Ferric neuroglobin is slowly reduced by H 2 S and catalyzes its inefficient oxidative conversion to thiosulfate. Mutation of the distal His 64 residue to alanine promotes rapid binding of H 2 S and its efficient conversion to oxidized products. X-ray absorption, EPR, and resonance Raman spectroscopy highlight the chemically different reaction options influenced by the distal histidine ligand. This study provides mechanistic insights into how the distal heme ligand in neuroglobin caps its reactivity toward H 2 S and identifies by cryo-mass spectrometry a range of sulfide oxidation products with 2-6 catenated sulfur atoms with or without oxygen insertion, which accumulate in the absence of the His 64 ligand.Neuroglobin is a member of the globin family (1) that is expressed primarily in neuronal tissues (2) but also in other metabolically highly active endocrine tissues, such as the adrenal gland and the pancreatic islets of Langerhans (3, 4). Although the physiological role of neuroglobin is still elusive, early studies suggested that it might be involved in oxygen supply (1, 5). However, neuroglobin is generally expressed at low levels and exhibits a high autoxidation rate, producing reactive oxygen species, which makes its role as an O 2 carrier unlikely (6). Neuroglobin can also scavenge reactive oxygen species and reduce nitrite to NO under hypoxic conditions (7-9).In contrast to hemoglobin and myoglobin, two histidine residues coordinate the heme cofactor in neuroglobin in the ferrous and ferric states (Fig. 1A). The crystal structure of neuroglobin reveals a high structural similarity to myoglobin, despite Ͻ25% amino acid sequence similarity between the proteins. The structure reveals a large hydrophobic cavity, which connects the proximal and distal sides of the heme (7). His 64 and His 96 coordinate the heme on the distal and proximal sides, respectively (10). Exogenous ligands like O 2 , CO, and NO bind to ferrous neuroglobin (Fe II -Ngb) 2 on the distal side, and binding is limited by dissociation of His 64 , which is slow (6,(11)(12)(13)(14). Limited characterization of the binding of sulfide to ferric neuroglobin (Fe III -Ngb) has...

The multifunctional protein p53 is the central molecular sensor of cellular stresses. The canonical function of p53 is to transcriptionally activate target genes in response to, for example, DNA damage that may trigger apoptosis. Recently, p53 was also found to play a role in the regulation of necrosis, another type of cell death featured by the mitochondrial permeability transition (mPT). In this process, p53 directly interacts with the mPT regulator cyclophilin D, the detailed mechanism of which however remains poorly understood. Here, we report a comprehensive computational investigation of the p53−cyclophilin D interaction using molecular dynamics simulations and associated analyses. We have identified the specific cyclophilin D binding site on p53 that is located at proline 151 in the DNA binding domain. As a peptidyl-prolyl isomerase, cyclophilin D binds p53 and catalyzes the cis−trans isomerization of the peptide bond preceding proline 151. We have also characterized the effect of such an isomerization and found that the p53 domain in the cis state is overall more rigid than the trans state except for the local region around proline 151. Dynamical changes upon isomerization occur in both local and distal regions, indicating an allosteric effect elicited by the isomerization. We present potential allosteric communication pathways between proline 151 and distal sites, including the DNA binding surface. Our work provides, for the first time, a model for how cyclophilin D binds p53 and regulates its activity by switching the configuration of a specific site.

Mitochondria are the powerhouse of a cell, whose disruption due to mitochondrial pore opening can cause cell death, leading to necrosis and many other diseases. The peptidyl-prolyl cis–trans isomerase cyclophilin D (CypD) is a key player in the regulation of the mitochondrial pore. The activity of CypD can be modulated by the post-translational modification (PTM). However, the detailed mechanism of this functional modulation is not well understood. Here, we investigate the catalytic mechanism of unmodified and modified CypD by calculating the reaction free energy profiles and characterizing the function-related conformational dynamics using molecular dynamics simulations and associated analyses. Our results show that unmodified and modified CypD considerably lower the isomerization free energy barrier compared to a free peptide substrate, supporting the catalytic activity of CypD in the simulation systems. The unmodified CypD reduces the free energy difference between the cis and trans states of the peptide substrate, suggesting a stronger binding affinity of CypD toward cis, consistent with experiments. In contrast, phosphorylated CypD further stabilizes trans, leading to a lower catalytic rate in the trans-to-cis direction. The differential catalytic activities of the unmodified and phosphorylated CypD are due to a significant shift of the conformational ensemble upon phosphorylation under different functional states. Interestingly, the local flexibility is both reduced and enhanced at distinct regions by phosphorylation, which is explained by a “seesaw” model of flexibility modulation. The allosteric pathway between the phosphorylation site and a distal site displaying substantial conformational changes upon phosphorylation is also identified, which is influenced by the presence of the substrate or the substrate conformation. Similar conclusions are obtained for the acetylation of CypD using the same peptide substrate and the influence of substrate sequence is also examined. Our work may serve as the basis for the understanding of other PTMs and PTM-initiated allosteric regulations in CypD.