An Alternative Mechanism of Bioluminescence Color Determination in Firefly Luciferase
详细信息 查看全文
- 作者：Bruce R. Branchini ; Tara L. Southworth ; Martha H. Murtiashaw ; Rachelle A. Magyar ; Susan A. Gonzalez ; Maria C. Ruggiero ; and Justin G. Stroh
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：June 15, 2004
- 年：2004
- 卷：43
- 期：23
- 页码：7255 - 7262
- 全文大小：259K
- 年卷期：v.43,no.23(June 15, 2004)
- ISSN：1520-4995
文摘
Beetle luciferases (including those of the firefly) use the same luciferin substrate to naturallydisplay light ranging in color from green (
lambda.gif" BORDER=0 >max ~530 nm) to red (lambda.gif" BORDER=0 >max ~635 nm). In a recent communication,we reported (Branchini, B. R., Murtiashaw, M. H., Magyar, R. A., Portier, N. C., Ruggiero, M. C., andStroh, J. G. (2002) J. Am. Chem. Soc. 124, 2112-2113) that the synthetic adenylate of firefly luciferinanalogue D-5,5-dimethylluciferin was transformed into the emitter 5,5-dimethyloxyluciferin in bioluminescence reactions catalyzed by luciferases from Photinus pyralis and the click beetle Pyrophorusplagiophthalamus. 5,5-Dimethyloxyluciferin is constrained to exist in the keto form and fluoresces mainlyin the red. However, bioluminescence spectra revealed that green light emission was produced by thefirefly enzyme, and red light was observed with the click beetle protein. These results, augmented withsteady-state kinetic studies, were taken as experimental support for mechanisms of firefly bioluminescencecolor that require only a single keto form of oxyluciferin. We report here the results of mutagenesisstudies designed to determine the basis of the observed differences in bioluminescence color with theanalogue adenylate. Mutants of P. pyralis luciferase putative active site residues Gly246 and Phe250, aswell as corresponding click beetle residues Ala243 and Ser247 were constructed and characterized usingbioluminescence emission spectroscopy and steady state kinetics with adenylate substrates. Based on ananalysis of these and recently reported (Branchini, B. R., Southworth, T. L., Murtiashaw, M. H., Boije,H., and Fleet, S. E. (2003) Biochemistry 42, 10429-10436) data, we have developed an alternativemechanism of bioluminescence color. The basis of the mechanism is that luciferase modulates emissioncolor by controlling the resonance-based charge delocalization of the anionic keto form of the oxyluciferinexcited state.
lambda.gif" BORDER=0 >max ~530 nm) to red (lambda.gif" BORDER=0 >max ~635 nm). In a recent communication,we reported (Branchini, B. R., Murtiashaw, M. H., Magyar, R. A., Portier, N. C., Ruggiero, M. C., andStroh, J. G. (2002) J. Am. Chem. Soc. 124, 2112-2113) that the synthetic adenylate of firefly luciferinanalogue D-5,5-dimethylluciferin was transformed into the emitter 5,5-dimethyloxyluciferin in bioluminescence reactions catalyzed by luciferases from Photinus pyralis and the click beetle Pyrophorusplagiophthalamus. 5,5-Dimethyloxyluciferin is constrained to exist in the keto form and fluoresces mainlyin the red. However, bioluminescence spectra revealed that green light emission was produced by thefirefly enzyme, and red light was observed with the click beetle protein. These results, augmented withsteady-state kinetic studies, were taken as experimental support for mechanisms of firefly bioluminescencecolor that require only a single keto form of oxyluciferin. We report here the results of mutagenesisstudies designed to determine the basis of the observed differences in bioluminescence color with theanalogue adenylate. Mutants of P. pyralis luciferase putative active site residues Gly246 and Phe250, aswell as corresponding click beetle residues Ala243 and Ser247 were constructed and characterized usingbioluminescence emission spectroscopy and steady state kinetics with adenylate substrates. Based on ananalysis of these and recently reported (Branchini, B. R., Southworth, T. L., Murtiashaw, M. H., Boije,H., and Fleet, S. E. (2003) Biochemistry 42, 10429-10436) data, we have developed an alternativemechanism of bioluminescence color. The basis of the mechanism is that luciferase modulates emissioncolor by controlling the resonance-based charge delocalization of the anionic keto form of the oxyluciferinexcited state.