Active site hydrophobicity is critical to the bioluminescence activity of Vibrio harveyi luciferase

Vibrio harveyi luciferase is an αβ heterodimer containing a single active site, proposed earlier to be at a cleft in the a subunit. In this work, six conserved phenylalanine residues at this proposed active site were subjected to site-directed mutations to investigate their possible functional roles and to delineate the makeup of luciferase active site. After initial screening of Phe - Ala mutants, αF46, αF49, αF114, and αF117 were chosen for additional mutations to Asp, Ser, and Tyr. Comparisons of the general kinetic properties of wild-type and mutated luciferases indicated that the hydrophobic nature of αF46, αF49, αF114, and αF117 was important to luciferase V m a x and V m a x /K m , which were reduced by 3-5 orders of magnitude for the Phe - Asp mutants. Both aF46 and αF117 also appeared to be involved in the binding of reduced flavin substrate. Additional studies on the stability and yield of the 4a-hydroperoxyflavin intermediate II and measurements of decanal substrate oxidation by aF46D, aF49D, αF114D, and αF117D revealed that their marked reductions in the overall quantum yield () were a consequence of diminished yields of luciferase intermediates and, with the exception of αF114D, emission quantum yield of the excited emitter due to the replacement of the hydrophobic Phe by the anionic Asp. The locations of these four critical Phe residues in relation to other essential and/or hydrophobic residues are depicted in a refined map of the active site. Functional implications of these residues are discussed.