Defining the structure of ferryl heme

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Metal-catalysed activation of oxygen is a cornerstone of biology. It is usually achieved using metal ions, typically either copper or iron. In the case of iron, there are both non-heme-containing and heme-containing enzymes which activate oxygen. This application focuses on the latter. The mechanism of activation involves formation of the iconic Compound I and Compound II intermediates, which are used across a diverse group of catalytic heme enzymes that include all the cytochrome P450s (involved in drug metabolism), the nitric oxide synthases (involved in cell signalling), and the terminal oxidases (involved in respiration), plus the heme dioxygenases (involved in formation of NAD) and heme peroxidases (involved in peroxide detoxification). Compounds I and II are such crucial intermediates in so many processes that defining their structure and understanding their reactivity, and in particular their protonation states, has been a key question in the field over several decades. We intend to use neutron crystallography to decisively advance the field in this area. We intend to obtain neutron crystal structures of Compound II, and we propose three strategies to deliver this. This will identify the locations of all the protons in the Compound II intermediate and will allow us to make mechanistic comparisons across the family of heme enzymes. These are ambitious, technically difficult and time-consuming experiments. But the likely gains are very high, because we have chance to finally resolve questions that have eluded the whole community for many years.