PDF(Pubmed)
Abstract：
The diflavin NADPH-cytochrome P450 reductase (CYPOR) plays a critical role in human cytochrome P450 (CYP) activity by sequentially delivering two electrons from NADPH to CYP enzymes during catalysis. Although electron transfer to forty-eight human CYP enzymes by the FMN hydroquinone of CYPOR is well-known, the role of the linker between the NH2-terminus membrane-binding domain (MBD) and FMN domain in supporting the activity of P450 enzymes remains poorly understood. Here we demonstrate that a linker with at least eight residues is required to form a functional CYPOR-CYP2B4 complex. The linker has been shortened in two amino-acid increments from Phe44 to Ile57 using site directed mutagenesis. The ability of the deletion mutants to support cytochrome P450 2B4 (CYP2B4) catalysis and reduce ferric CYP2B4 was determined using an in vitro assay and stopped-flow spectrophotometry. Steady-state enzyme kinetics showed that shortening the linker by 8-14 amino acids inhibited (63-99%) the ability of CYPOR to support CYP2B4 activity and significantly increased the Km of CYPOR for CYP2B4. In addition, the reductase mutants decreased the rate of reduction of ferric CYP2B4 (46-95%) compared to wildtype when the linker was shortened by 8-14 residues. These results indicate that a linker with a minimum length of eight residues is necessary to enable the FMN domain of reductase to interact with CYP2B4 to form a catalytically competent complex. Our study provides evidence that the length of the MBD-FMN domain linker is a major determinant of the ability of CYPOR to support CYP catalysis and drug metabolism by P450 enzymes. PREAMBLE: This manuscript is dedicated in memory of Dr. James R. Kincaid who was the doctoral advisor to Dr. Freeborn Rwere and a longtime collaborator and friend of Dr. Lucy Waskell. Dr. James R. Kincaid was a distinguished professor of chemistry specializing in resonance Raman (rR) studies of heme proteins. He inspired Dr. Rwere (a Zimbabwean native) and three other Zimbabweans (Dr. Remigio Usai, Dr. Daniel Kaluka and Ms. Munyaradzi E. Manyumwa) to use lasers to document subtle changes occurring at heme active site of globin proteins (myoglobin and hemoglobin) and cytochrome P450 enzymes. Dr. Rwere appreciate his contributions to the development of talented Black scientists from Africa.
摘要：
二黄素NADPH-细胞色素P450还原酶（CYPOR）通过在催化过程中从NADPH顺序传递两个电子到CYP酶，在人细胞色素P450（CYP）活性中起着关键作用。尽管CYPOR的FMN氢醌对48种人CYP酶的电子转移是众所周知的，NH2-末端膜结合域(MBD)和FMN域之间的连接子在支持P450酶活性方面的作用仍知之甚少.这里我们证明了具有至少八个残基的接头是形成功能性CYPOR-CYP2B4复合物所必需的。使用定点诱变将接头从Phe44缩短到Ile57的两个氨基酸增量。使用体外测定和停流分光光度法确定缺失突变体支持细胞色素P4502B4（CYP2B4）催化和还原CYP2B4铁的能力。稳态酶动力学表明，将接头缩短8-14个氨基酸会抑制（63-99％）CYPOR支持CYP2B4活性的能力，并显着增加CYP2B4的CYPORKm。此外,当接头缩短8-14个残基时，与野生型相比，还原酶突变体降低了CYP2B4铁的还原率（46-95％）。这些结果表明,具有8个残基的最小长度的接头是使还原酶的FMN结构域能够与CYP2B4相互作用以形成催化能力复合物所必需的。我们的研究提供了证据，表明MBD-FMN结构域接头的长度是CYPOR支持P450酶的CYP催化和药物代谢能力的主要决定因素。PREAMBLE：本手稿专门纪念JamesR.Kincaid博士，他是FreebornRwe博士的博士顾问，也是LucyWaskell博士的长期合作者和朋友。JamesR.Kincaid博士是一位杰出的化学教授，专门研究血红素蛋白的共振拉曼（rR）研究。他启发了Rwe博士（津巴布韦人）和其他三个津巴布韦人（RemigioUsai博士，DanielKaluka博士和MunyaradziE.Manyumwa女士）使用激光记录珠蛋白（肌红蛋白和血红蛋白）和细胞色素P450酶的血红素活性位点发生的细微变化。Rwere博士赞赏他对非洲才华横溢的黑人科学家的发展做出的贡献。
