MECHANISM OF CYTOCHROME OXIDASE

SIR提供d-[“N]氨基酮丙酸； L-[环_2羟基酪氨酸；0.5g D，L-[环-N]组氨酸；0.5G本项目的目的是 利用傅里叶变换红外差示光谱鉴定特定氨基酸 它们在质子泵的机制中起着重要的作用 血红素-铜呼吸氧化酶。这是一个酶的大家族， 包括细胞色素氧化酶，利用产生的自由能 可通过将氧气还原为水来产生电压 以及生物膜上的pH梯度。不同的状态 作为催化循环中重要中间体的酶可以 在实验室中产生，FTIR差示光谱可以 检测这些状态之间的差异。这项技术非常 敏感的，因此单一氨基的质子化或去质子化 蛋白质中的酸可以作为FTIR差异的变化来检测 频谱。确定蛋白质中的氨基酸是 负责观察到的FTIR光谱变化需要使用 稳定的同位素被结合到酶中。质量的变化 导致振动吸收带发生可解释的移动 由FTIR监测。标记的组氨酸和酪氨酸已经被 结合到细菌血红素-铜氧化酶中，细胞色素bo3来自 大肠埃希菌。目前，我们正在检测含有以下成分的纯化酶 傅里叶变换红外光谱分析同位素标记氨基酸 与综合光谱学实验室的小组合作 在LANL。

The SIR provided d-["N]Aminolevulinic Acid.HC I; 7.07g L-[ring_2 HdTyrosine; 0.5g D,L-[ring- N2]Histidine; 0.5g The purpose of this project is to utilize FTIR difference spectroscopy to identify specific amino acids that have important roles in the mechanism of proton pumping of the heme-copper respiratory oxidases. This is a large family of enzymes, including cytochrome oxidase, that utilize the free energy made available from the reduction of dioxygen to water to make a voltage and pH gradients across the biological membrane. Different states of the enzyme that are important intermediates in the catalytic cycle can be generated in the laboratory, and FTIR difference spectroscopy can detect differences between these states. The technique is very sensitive, so that the protonation or deprotonation of a single amino acid in the protein can be detected as a change in the FTIR difference spectrum. Identifying the amino acids in the protein that are responsible for observed changes in the FTIR spectrum requires the use of stable isotopes incorporated in the enzyme. The change in mass results in interpretable shifts in the vibrational absorption bands monitored by FTIR. The labeled histidine and tyrosine have been incorporated into a bacterial heme-copper oxidase, cytochrome bo3 from E. coli. Currently, we are examining the purified enzymes containing the isotopically labeled amino acids using FTIR spectroscopy in collaboration with the group at the Integrated Spectroscopy Laboratory at LANL.