Direct Infrared Detection of the Covalently Ring Linked His-Tyr Structure in the Active Site of the Heme-Copper Oxidases
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- 作者：Farol Tomson ; James A. Bailey ; Robert B. Gennis ; Clifford J. Unkefer ; Zizhong Li ; Louis A. Silks ; Rodolfo A. Martinez ; Robert J. Donohoe ; R. Brian Dyer ; and William H. Woodruff
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：December 3, 2002
- 年：2002
- 卷：41
- 期：48
- 页码：14383 - 14390
- 全文大小：118K
- 年卷期：v.41,no.48(December 3, 2002)
- ISSN：1520-4995
文摘
Infrared spectroscopy, isotopic labeling ([15N
,
n.gif" BORDER=0 >]histidine and ring-deuterated tyrosine), syntheticmodel studies, and normal mode calculations are employed to search for the spectroscopic signatures ofthe unique, covalently linked (His Nn.gif" BORDER=0 >-Cn.gif" BORDER=0 > Tyr) biring structure in the heme-copper oxidases. The specificenzyme examined is the cytochrome bo3 quinol oxidase of E. coli. Infrared features of histidine andtyrosine are identified in the frequency regions of imidazole and phenol ring stretching modes (1350-1650 cm-1) and C-H and N-H stretching modes as well as overtones and combinations (>3000 cm-1).Two of these, at ca. 1480 and 1550 cm-1, and their combination tones between 3010 and 3040 cm-1, aredefinitively identified with the biring structure involving H284 and Y288 in the E. coli enzyme. Studiesof a synthetic analogue of the H-Y structure, 4-methylimidazole covalently linked to p-cresol, show thata feature near 1540 cm-1 is unique to the biring structure and is absent from the infrared spectrum of4-methylimidazole or p-cresol alone. This feature is readily detectable by infrared difference techniques,and offers a direct spectroscopic probe for potential radical production involving the H-Y structure inthe O2 reduction cycle of the oxidases.
,n.gif" BORDER=0 >]histidine and ring-deuterated tyrosine), syntheticmodel studies, and normal mode calculations are employed to search for the spectroscopic signatures ofthe unique, covalently linked (His Nn.gif" BORDER=0 >-Cn.gif" BORDER=0 > Tyr) biring structure in the heme-copper oxidases. The specificenzyme examined is the cytochrome bo3 quinol oxidase of E. coli. Infrared features of histidine andtyrosine are identified in the frequency regions of imidazole and phenol ring stretching modes (1350-1650 cm-1) and C-H and N-H stretching modes as well as overtones and combinations (>3000 cm-1).Two of these, at ca. 1480 and 1550 cm-1, and their combination tones between 3010 and 3040 cm-1, aredefinitively identified with the biring structure involving H284 and Y288 in the E. coli enzyme. Studiesof a synthetic analogue of the H-Y structure, 4-methylimidazole covalently linked to p-cresol, show thata feature near 1540 cm-1 is unique to the biring structure and is absent from the infrared spectrum of4-methylimidazole or p-cresol alone. This feature is readily detectable by infrared difference techniques,and offers a direct spectroscopic probe for potential radical production involving the H-Y structure inthe O2 reduction cycle of the oxidases.