RUI: Characterization of Reactive Intermediates in the Cytochrome P450 Pathway

该奖项在无机、生物无机和有机金属化学项目中获得，支持北卡罗来纳大学格林斯博罗分校的Gregory Raner教授利用快速混合和数据采集技术，直接观察和表征细胞色素P450两种不同催化途径中的中间体。第一个目标是生成P450BM3-F87G与氧原子供体反应的两个相关中间体并对其进行光谱表征。第二个目标是鉴定P450BM3-F87G过氧通路中的瞬时中间体。用于快速混合和监测酶的光谱特性的停流和冻灭EPR方法将用于实现这两个特定目标。将氧原子供体、间氯过氧苯甲酸和碘索苯与P450BM3的F87G突变体结合，在几毫秒到几分钟的时间内，将获得光谱。此外，混合后的样品将在不同的时间点快速冷冻，并使用电子顺磁共振波谱分析，以提供与反应中形成的中间体身份相关的支持性实验细节。将制备反应混合物，其中醛底物将在过氧化氢存在下与P450BM3-F87G反应，导致醛的去甲酰基化，酶中的血红素辅因子进行化学修饰。类似的冻灭EPR实验也将进行。研究目标的成功实现将为细胞色素P450的催化机制以及电子结构和活性之间的关系提供关键细节，这可能会导致细胞色素P450酶在生物技术中的更有效利用。该研究将使本科生和硕士生具备化学和生物学界面的宝贵研究技能。

This award in the Inorganic, Bioinorganic and Organometallic Chemistry program supports Professor Gregory Raner at the University of North Carolina at Greensboro to directly observe and characterize intermediates in two distinct catalytic pathways of cytochrome P450 by taking advantage of rapid mixing and data acquisition techniques. The first objective is to generate and spectrally characterize two related intermediates in the reaction of P450BM3-F87G with oxygen atom donors. The second objective is to identify transient intermediates in the peroxo-pathway for P450BM3-F87G. The stopped-flow and freeze-quench EPR methods for rapid mixing and monitoring of the spectroscopic properties of the enzyme will be used to achieve both specific objectives. The oxygen atom donors, m-chloroperoxybenzoic acid and iodosobenzene will be combined with the F87G mutant of P450BM3 and over the time course of several milliseconds to several minutes, spectra will be acquired. In addition, samples will be rapidly frozen at various time points after mixing, and analyzed using electron paramagnetic resonance spectroscopy in order to provide supporting experimental details relating to the identity of intermediates formed in the reactions. Reaction mixtures will be prepared in which an aldehyde substrate will react with P450BM3-F87G in the presence of hydrogen peroxide, resulting in deformylation of the aldehyde, with chemical modification of the heme cofactor in the enzyme. Analogous freeze-quench EPR experiments will also be carried out. Successful attainment of the research objectives will provide critical details concerning the catalytic mechanism of cytochrome P450 concerning relationships between electronic structure and activity, which may lead to the more efficient use of cytochrome P450 enzymes in biotechnology. The proposed study will equip undergraduate and Masters students with the valuable research skills at the interface of chemistry and biology.