X-RAY ABSORPTION SPECTROSCOPY OF CHLOROPEROXIDASE COMPOUND II

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 以前的EXAFS研究报告说，巯基连接的铁（IV）氧代（ferryl）在氯过氧化物酶化合物II的物种是质子化在pH值为6.5。这一观察结果的重要性在于它与C-H键活化的联系。金属氧合物提取氢的能力与形成的O-H键的强度成比例;在氧化血红素酶中，这取决于化合物I（铁基自由基物种）的还原电位和化合物II（铁基物种）的pKa。随后的氯过氧化物酶化合物II的穆斯堡尔测量揭示了存在两个不同的铁物种在一个。70：30的比例。我们发现组分浓度的70：30比例与pH无关，并且我们用不同的氧化剂（过氧化物和过氧乙酸）和还原剂（抗坏血酸盐和对苯酚磺酸）获得了相同的70：30比例。最近，我们已经确定了制备CPO-II的单一组分的方法。CPO与间氯过苯甲酸和抗坏血酸的反应导致单一的铁基物种。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，我们可以选择性地获得CPO-II的主要成分，通过化学修饰的氯过氧化物酶。CPO与焦碳酸二乙酯的反应修饰组氨酸残基。其中一个His残基位于CPO活性位点附近的质子穿梭中。改性的CPO与过氧乙酸和抗坏血酸盐的反应产生一种物质（。90%产率），穆斯堡尔参数与m-CPBA中间体和CPO-II的主要组分测量的参数几乎相同。我们提出了一个EXAFS检查的中间体制备1）反应的m-CPBA与CPO和2）化学修饰的质子穿梭。我们试图确定这些物种是质子化的Fe（IV）oxos。这些测量将提供洞察自然的巯基连接在其羟化血红素酶的使用。

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.