Nitroxyl (HNO) is a redox sibling of nitric oxide (NO) that targets distinct signalling pathways with pharmacological endpoints of high significance in the treatment of heart failure. Beneficial HNO effects depend, in part, on its ability to release calcitonin gene-related peptide (CGRP) through an unidentified mechanism. Here we propose that HNO is generated as a result of the reaction of the two gasotransmitters NO and H₂S. We show that H₂S and NO production colocalizes with transient receptor potential channel A1 (TRPA1), and that HNO activates the sensory chemoreceptor channel TRPA1 via formation of amino-terminal disulphide bonds, which results in sustained calcium influx. As a consequence, CGRP is released, which induces local and systemic vasodilation. H₂S-evoked vasodilatatory effects largely depend on NO production and activation of HNO–TRPA1–CGRP pathway. We propose that this neuroendocrine HNO–TRPA1–CGRP signalling pathway constitutes an essential element for the control of vascular tone throughout the cardiovascular system.

The acid-base behavior of amino acids is an important subject of study due to their prominent role in enzyme catalysis, substrate binding and protein structure. Due to interactions with the protein environment, their pK_as can be shifted from their solution values and, if a protein has two stable conformations, it is possible for a residue to have different “microscopic”, conformation-dependent pK_a values. In those cases, interpretation of experimental measurements of the pK_a is complicated by the coupling between pH, protonation state and protein conformation. We explored these issues using Nitrophorin 4 (NP4), a protein that releases NO in a pH sensitive manner. At pH 5.5 NP4 is in a closed conformation where NO is tightly bound, while at pH 7.5 Asp30 becomes deprotonated, causing the conformation to change to an open state from which NO can easily escape. Using constant pH molecular dynamics we found two distinct microscopic Asp30 pK_as: 8.5 in the closed structure and 4.3 in the open structure. Using a four-state model, we then related the obtained microscopic values to the experimentally observed “apparent” pK_a, obtaining a value of 6.5, in excellent agreement with experimental data. This value must be interpreted as the pH at which the closed to open population transition takes place. More generally, our results show that it is possible to relate microscopic structure dependent pKa values to experimentally observed ensemble dependent apparent pK_as and that the insight gained in the relatively simple case of NP4 can be useful in several more complex cases involving a pH dependent transition, of great biochemical interest. The interaction of an amino acid with its protein environment can result in an acid-base behavior that is very different from what would be observed in solution. This environment can be greatly altered when the protein changes conformation. As a result, the amino acid will have two different “microscopic” pK_a values. Nitrophorin 4 is a good case study to explore this behavior, because it undergoes a pH-dependent conformational change that is well characterized experimentally. Using computer simulation tools, we found that the key titratable Aspartic acid 30, has two very different microscopic pK_as: 4.3 and 8.5, which are significantly different to the observed transition pK_a in solution. However, using a simple model, we were able to understand how this causes the conformational change to take place at pH∼6.5, as measured experimentally. The insight gained in this relatively simple case can be useful in other more complex cases where the apparent pK_a is also a result of the interplay of different conformations where some amino acids experience very different environments.

In contrast to the wide spectrum of cytochrome P450 monooxygenases, there are only 2 heme-based dioxygenases in humans: tryptophan dioxygenase (hTDO) and indoleamine 2,3-dioxygenase (hIDO). hTDO and hIDO catalyze the same oxidative ring cleavage reaction of L-tryptophan to N-formyl kynurenine, the initial and rate-limiting step of the kynurenine pathway. Despite immense interest, the mechanism by which the 2 enzymes execute the dioxygenase reaction remains elusive. Here, we report experimental evidence for a key ferryl intermediate of hIDO that supports a mechanism in which the 2 atoms of dioxygen are inserted into the substrate via a consecutive 2-step reaction. This finding introduces a paradigm shift in our understanding of the heme-based dioxygenase chemistry, which was previously believed to proceed via simultaneous incorporation of both atoms of dioxygen into the substrate. The ferryl intermediate is not observable during the hTDO reaction, highlighting the structural differences between the 2 dioxygenases, as well as the importance of stereoelectronic factors in modulating the reactions.