The recently proposed model for the bacteria luciferase-flavin mononucleotide complexidentifies a number of critical intermolecular interactions that define a binding platform for the isoalloxazinering of flavin [Lin, L. Y., Sulea, T., Szittner, R., Vassilyev, V., Purisima, E. O., and Meighen, E. A.(2001)
Protein Sci. 10, 1563-1571]. A key interaction involving van der Waals contact between theisopropyl side chain of
Val173 and the 7,8-dimethyl benzene plane of the isoalloxazine chromophorerepresents an important target to test the validity of the proposed model. Here, structure-function analysisof luciferase variants carrying single point mutations at position
173 have verified the functional layoutof the active site architecture and implicated this site directly in flavin binding. Moreover, a decrease inthe stability of the enzyme-bound C4a-hydroperoxyflavin intermediate in the mutants could account forchanges in saturation with the fatty aldehyde substrate. A predicted red-shift on mutation of position
173 to increase its polarity confirmed that
Val173 was an integral component of the chromophore-binding microenvironment. Introduction of mutations in residues that contact the pyrimidine plane of theisoalloxazine chromophore (
A75G/C106V) into the
V173A,
V173C,
V173T, and
V173S mutantsled to the retention of high levels of enzyme activity (10-40% of wild type) and further red-shifted theemission spectra in the triple mutants. The additivity of the mutation-induced red-shifts in the emissionwavelength spectrum provides the basis toward engineering luciferase variants that emit different lightcolors with the proposed flavin-luciferase model complex as a design reference.