Nature’s evolutionary journey is defined by exceptional efficiency, with each adaptation fine‑tuned to minimise waste and optimise resource use. Enzymes lie at the heart of this process: biological catalysts accelerate chemical transformations with remarkable specificity. These molecular machines underpin vital functions, ranging from nutrient breakdown to synthesising essential biomolecules, thereby conserving energy and maintaining the equilibrium of natural systems. Among the most striking illustrations of enzymatic prowess are nitrogenases, a superfamily present across numerous organisms. Over roughly 3.8 billion years of evolution, nitrogenases have evolved the ability to cleave extraordinarily stable bonds, such as those in nitrogen (N2) and hydrogen (H2), under mild conditions. This capability makes them inspiring models for designing of new sustainable biotechnologies. Structurally, nitrogenases comprise two main components: a reductase and a catalytic unit, each harbouring intricate, oxygen-sensitive metal clusters that sustain the ATP-dependent electron transfer mechanism. This family's most extensively characterised member is the molybdenum nitrogenase (Mo‑N2ase), which is responsible for N2-fixation to produce ammonia (NH3). Yet phylogenetic studies reveal related reductases in anaerobic prokaryotes, participating in processes like bacterial photosynthesis and archaeal methanogenesis.
This thesis focuses on a nitrogenase-like Dark‑Operative Protochlorophyllide Oxidoreductase (DPOR). DPOR reduces the C17=C18 double bond of protochlorophyllide (Pchlide) to yield chlorophyllide (Chlide). It consists of BchL (a homodimer with two ATP‑binding sites and a shared [4Fe–4S] cluster) and BchNB (an (αβ)2 heterotetramer containing both a [4Fe–4S] cluster and a Pchlide‑binding site). Transient association of these components, driven by ATP hydrolysis, mediates sequential electron flow from BchL to BchNB and ultimately to Pchlide.Despite decades of study, key aspects of nitrogenase catalysis remain unresolved: for example the order of events taking place at any transient association, the function of the (αβ)₂ architecture and its degree of cooperativity during the mechanism, and the role of ATP, which is not totally clear. DPOR is largely unexplored and its thermodynamic properties, such as reduction potential of iron-sulfur clusters have not been reported yet. The key adavntages in the study of DPOR, compared to related enzymes, is that both its substrate and product absorb visible light.
Built on this advantage, in this thesis, DPOR has been investigated via visible‑spectroscopy, alone and coupled with electrochemistry. This thesis explores the use of an alternative electron donor for DPOR. It presents the results obtained to validate electrochemical and spectroelectrochemical methods, including preliminary data related to using spectroelectrochemistry to measure the reduction potential of BchL. It also introduces a novel direction in the study: the observation of the Pchlide: BchNB complex (Enzyme-Substrate (ES) complex) via visible spectroscopy. This rare spectroscopic feature was exploited to monitor enzyme kinetics and develop a method for analysing the associated data and let to the study of coopertivity during ES-complex formation.
