Alcohol dehydrogenase from the thermoacidophilic Sulfolobus solfataricus (SsADH) is a thermo-philic NAD + -dependent homotetrameric zinc enzyme whose crystal structure has been recently determined at 1.85 Å resolution by using a selenomethionine-substituted enzyme. In this report the steady-state fluorescence properties of SsADH are related to the two fluorophores Trp95 and 117 located inside the monomer structure and whose indole ring centers are at 5.7 A ° to each other. The relatively blue emission of the enzyme (lmax 5 320 nm) is due to the highly hydrophobic character of the microenvironment determined by six and seven non-polar residues in close contact (,7 A ° ) with Trp95 and 117, respectively. However, the contribution of the two residues to intrinsic fluores-cence appears different since the Trp95 and 117 indole rings are found in the vicinity of five and one polar residue side chains, respectively. The fluorescence intensity of the selenomethionine-substituted enzyme is found to be 40% lower than that of the natural enzyme. Moreover, four out of the nine methionine residues per monomer are found in the vicinity (,6 A ° ) of as many tyrosine residue side chains, while no methionine-tryptophan interaction is present in the structure. Presum-ably, selenium acts as a quencher of the nearby tyrosine emission more efficiently than does sulfur, due to its larger electron cloud and polarizability. It cannot be excluded that an effect of selenium is to stabilize the tyrosinate ion allowing a more extended delocalization of the negative charge.Therefore, the decreased tryptophan emission of the seleno-protein would reflect the lower quantum yield of the tyrosine in its ionized state.
Steady-state fluorescence properties of Sulfolobus solfataricus alcohol dehydrogenase and its selenomethionyl derivative.
Raia CA
2003
Abstract
Alcohol dehydrogenase from the thermoacidophilic Sulfolobus solfataricus (SsADH) is a thermo-philic NAD + -dependent homotetrameric zinc enzyme whose crystal structure has been recently determined at 1.85 Å resolution by using a selenomethionine-substituted enzyme. In this report the steady-state fluorescence properties of SsADH are related to the two fluorophores Trp95 and 117 located inside the monomer structure and whose indole ring centers are at 5.7 A ° to each other. The relatively blue emission of the enzyme (lmax 5 320 nm) is due to the highly hydrophobic character of the microenvironment determined by six and seven non-polar residues in close contact (,7 A ° ) with Trp95 and 117, respectively. However, the contribution of the two residues to intrinsic fluores-cence appears different since the Trp95 and 117 indole rings are found in the vicinity of five and one polar residue side chains, respectively. The fluorescence intensity of the selenomethionine-substituted enzyme is found to be 40% lower than that of the natural enzyme. Moreover, four out of the nine methionine residues per monomer are found in the vicinity (,6 A ° ) of as many tyrosine residue side chains, while no methionine-tryptophan interaction is present in the structure. Presum-ably, selenium acts as a quencher of the nearby tyrosine emission more efficiently than does sulfur, due to its larger electron cloud and polarizability. It cannot be excluded that an effect of selenium is to stabilize the tyrosinate ion allowing a more extended delocalization of the negative charge.Therefore, the decreased tryptophan emission of the seleno-protein would reflect the lower quantum yield of the tyrosine in its ionized state.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
