Ser was replaced at position 286 of firefly luciferase (Luciola mingrelica) by a series of naturally occurring and unnatural amino acids, The effect of these substitutions on the properties of luciferase, such as thermostability, pH dependence, and color of light emitted, was investigated. For these purposes, the Ser286 codon (AGT)was replaced by an amber stop codon (TAG) within the luciferase gene and transformed into Escherichia coli strains producing specific amber suppressor tRNA's to express luciferase with different substitutions at this position, The incorporation of Leu, Lys, Tyr, or Gin at this position reduced the thermostability of mutated luciferases. The color of emitted light changed upon substitution from yellow-green (lambda(max) 582 nm) for the wild-type enzyme having Ser286 to, for example, red (lambda(max) 622 nm) for luciferase having Leu286, For further evaluation of the structural relationship between the amino acid position at 286 and the wavelength of emitted light, we used the method of in vitro incorporation of unnatural amino acids, which involves readthrough of a nonsense (UAG) codon by a misacylated suppressor tRNA, The amino acids incorporated at position 286 in this fashion included O-glucosylated serine, serine phosphonate, tyrosine phosphate, and tyrosine methylenephosphonate. The wavelength of light emitted by the luciferase analogues was measured, While the introduction of serine phosphonate and glucosylated serine did not change the lambda(max) of light produced by luciferase, the incorporation of tyrosine phosphate and tyrosine methylenephosphonate into position 286 altered the spectra of emitted light compared with those of Ser286 and Tyr286, The pH dependence of the wavelength of light emitted by the luciferases containing the negatively charged phosphorylated Tyr analogues was demonstrated and could be rationalized in terms of the pK(a)'s of the phosph(on)ate oxygens.