Role of the Luciferase C-Terminal Domain in Firefly Bioluminescence

. Luciferase consists of an N-terminal domain (N-domain) and a C-terminal domain (C-domain), which are linked by a flexible loop. Substrates bind mainly within the N-domain, suggesting that the N-domain alone should be capable of maintaining the BL. However, an experimental study indicated that when the C-domain of wild-type luciferase (WT) is removed, the truncated N-domain type luciferase (NT) retains only 0.03% of firefly BL activity compared with the WT system. This produces some fundamental questions: what is the role of the C-domain in firefly BL and why does the deletion of the C-domain cause such a drastic decrease in firefly BL activity? In this article, we address these questions in detail via multiscale theoretical calculations. Our results show that the C-domain plays a positive catalytic role by enhancing the affinity of substrates, promoting the formation of preorganized and compact conformations of critical intermediates, and sustaining the enzyme-generated internal electric field (IEF) favorable for the firefly BL reaction. In the absence of the C-domain, these BL-favorable factors are severely disrupted, leading to the suppression of the firefly BL efficiency. This study provides the first computational elucidation of the entire firefly BL process within a realistic protein environment and introduces the concept of IEF catalysis into the context of BL. Most importantly, we deeply identify and characterize the crucial role of the C-domain and some key residues (LYS439, LYS529, and ARG533) in firefly BL.