Effects of mutations of the alpha His45 residue of Vibrio harveyi luciferase on the yield and reactivity of the flavin peroxide intermediate.
This work was undertaken to investigate the functional consequences of mutations of the essential alpha His45 residue of Vibrio harveyi luciferase, especially with respect to the yield and reactivity of the flavin 4a-hydroperoxide intermediate II. A total of 14 luciferase variants, each with a different single-residue replacement for the alpha His45, were examined. These variants showed changes, mostly slight, in their light decay rates of the nonturnover luminescence reaction and in their Km values for decanal and reduced riboflavin 5'-phosphate (FMNH2). All alpha His45 mutants, however, showed markedly reduced bioluminescence activities, the magnitude of the reduction ranging from about 300-fold to 6 orders of magnitude. Remarkably, a good correlation was obtained for the wild-type luciferase, 12 alpha His45-mutated luciferases, and six additional variants with mutations of other alpha-subunit histidine residues between the degrees of luminescence activity reduction and the dark decay rates of intermediate II. Such a correlation further indicates that the activation of the O-O bond fission is an important function of the flavin 4a-hydroperoxide intermediate II. Both alpha H45G and alpha H45W were found to bind near-stoichiometric amounts of FMNH2. Moreover, each variant catalyzed the oxidation of bound FMNH2 by two mechanisms, with a minor pathway leading to the formation of a luminescence-active intermediate II and a major dark pathway not involving any detectable flavin 4a-hydroperoxide species. This latter pathway mimics that in the normal catalysis by flavooxidases, and its elicitation in luciferase was demonstrated for the first time by single-residue mutations.
Li
H
H
Department of Biology and Biochemistry, University of Houston, Texas 77204-5513, USA.
Ortego
B C
BC
Maillard
K I
KI
Willson
R C
RC
Tu
S C
SC
D000447:Aldehydes / Q000737:chemistry; D002384:Catalysis; D003903:Deuterium / Q000737:chemistry; D005486:Flavin Mononucleotide / Q000737:chemistry; D005415:Flavins / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry / Q000235:genetics*; D008156:Luciferases / Q000737:chemistry / Q000235:genetics*; D008163:Luminescent Measurements; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D013053:Spectrophotometry; D014733:Vibrio / Q000201:enzymology*
D000447:Aldehydes; D005415:Flavins; C064157:high fluorescent intermediate II; C021170:decanaldehyde; D006639:Histidine; D005486:Flavin Mononucleotide; D003903:Deuterium; D008156:Luciferases