X-RAY ABSORPTION SPECTROSCOPY OF CHLOROPEROXIDASE COMPOUND II

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 以前的EXAFS研究报告说，氯过氧化物酶II中的硫酸盐连接的Fe(IV)氧(铁)物种在pH 6.5下被质子化。这一观察的重要性在于它与C-H键的活化有关。在氧化型血红素酶中，这取决于化合物I(铁基物种)的还原潜力和化合物II(铁基物种)的pKa。随后对氯过氧化物酶化合物II的穆斯堡尔测量表明，AN中存在两个不同的铁基物种。70：30的比例。我们发现组分浓度的70：30的比例与pH无关，不同的氧化剂(过氧化氢和过氧乙酸)和还原剂(抗坏血酸和对苯酚磺酸)也获得了相同的70：30比例。最近，我们确定了一种制备CPO-II单一组分的方法。CPO与间氯苯甲酸和抗坏血酸反应生成单一的铁基物种。该中间体的穆斯堡尔参数与CPO-II的主要组分的穆斯堡尔参数相同。我们还发现，通过对氯过氧化物酶的化学修饰，可以选择性地获得CPO-II的主要成分。CPO与焦碳酸二乙酯的反应可修饰组氨酸残基。其中一个His残留物在CPO活动点附近的质子航天飞机中。改性的CPO与过氧乙酸和抗坏血酸反应生成一种(.产率为90%)，穆斯堡尔参数与间CPBA中间体和CPO-II的主要成分的测量参数几乎相同。我们建议用EXAFS分析1)间CPBA与CPO反应和2)对质子穿梭机进行化学修饰所得到的中间体。我们试图确定这些物种是否是质子化的Fe(IV)氧。这些测量将提供对大自然在其羟化血红素酶中使用硫酸盐连接的洞察。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A previous EXAFS investigation reported that the thiolate-ligated Fe(IV)oxo (ferryl) species in chloroperoxidase compound II is protonated at pH 6.5. The importance of this observation lies in its connection to C-H bond activation. The ability of metal-oxos to abstract hydrogen scales with the strength of the O-H bond formed; in oxidative heme enzymes, this depends on the reduction potential of compound I (a ferryl-radical species) and the pKa of compound II (a ferryl species). Subsequent Mossbauer measurements of chloroperoxidase compound II have revealed the presence of two distinct ferryl species in an . 70:30 ratio. We have found the 70:30 ratio of component concentrations to be independent of pH, and we have obtained the same 70:30 ratio with different oxidants (peroxide and peracetic acid) and reductants (ascorbate and p-phenolsulfonic acid). Recently, we have determined a means of preparing a single component of CPO-II. Reaction of CPO with meta-chloroperbenzoic acid and ascorbate results in a single ferryl species. The Mossbauer parameters of this intermediate are identical to those of the major component of CPO-II. We have also found that we can selectively access the major component of CPO-II through the chemical modification of chloroperoxidase. The reaction of CPO with diethylpyrocarbonate modifies histidine residues. One of these His residues is in a proton-shuttle near the CPO activesite. The reaction of modified CPO with peracetic acid and ascorbate yields a species (. 90% yield) with Mossbauer parameters that are virtually identical to the parameters measured for the m-CPBA intermediate and the major component of CPO-II. We propose an EXAFS examination of the intermediates prepared by 1) reaction of m-CPBA with CPO and 2) chemical modification of the proton shuttle. We seek to determine if these species are protonated Fe(IV)oxos. These measurements will provide insight into Nature's use of thiolate-ligation in its hydroxylating heme enzymes.