The aim of my PhD project was to obtain new structural and functional insights useful to draw a more detailed overall picture of the [FeFe]-hydrogenase maturation machinery. Indeed, although during recent years advances have been made in the knowledge of this maturation pathway, significant gaps remain in the understanding of how this process occurs.
In this context, my work has been developed in these topics:
The resolution of the tridimensional crystal structure of HydF, the key protein of the [FeFe]-hydrogenase maturation system. The results and the analysis of the structure and its domains are contained in Chapter 1 of the thesis. The obtained informations have also opened up new scenarios that have led me to investigate further aspects of the HydF protein structure-function relationship, reported in the other two chapters.
In the second Chapter I describe the work that has led to the characterization of the HydF FeS cluster binding pocket. In particular, we have analyzed the role, in the cluster coordination as well as in the hydrogenase activation, of two histidines present close to three cysteines all belonging to the highly conserved FeS cluster binding consensus sequence.
Finally, in the last part of my PhD work (whose results are collected in Chapter 3) I focused my attention on the biochemical characterization of the interactions between HydF and the other components of the [FeFe]-hydrogenase maturation process, which are needed for the activity of HydF both as a scaffold and a FeS cluster carrier in this pathway. Moreover, I investigated the HydF GTPase properties, which had been previously shown to be essential for the [FeFe]-hydrogenase activation
