Department of Anesthesiology, University of Michigan and VAMC, 2215 Fuller Road, Ann Arbor, MI, USA; Department of Anesthesiology, Perioperative and Pain Medicine, School of Medicine, Stanford University, 3174 Porter Dr, Palo Alto, CA, USA.
Department of Anesthesiology, University of Michigan and VAMC, 2215 Fuller Road, Ann Arbor, MI, USA.
J Inorg Biochem. 2024 Oct;259:112667. doi: 10.1016/j.jinorgbio.2024.112667. Epub 2024 Jul 16.
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 NH-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 K 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.
双黄素 NADPH-细胞色素 P450 还原酶（CYPOR）通过在催化过程中顺序将两个电子从 NADPH 递送至 CYP 酶，在人类细胞色素 P450（CYP）活性中发挥关键作用。尽管 FMN 氢醌将电子转移至四十八种人类 CYP 酶是众所周知的，但在支持 P450 酶活性方面，NH-末端膜结合结构域（MBD）和 FMN 结构域之间的连接体的作用仍知之甚少。在这里，我们证明至少需要 8 个残基的连接体才能形成功能性 CYPOR-CYP2B4 复合物。使用定点诱变，将连接体从 Phe44 到 Ile57 以两个氨基酸的增量缩短。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 P450 2B4（CYP2B4）催化和还原高铁细胞色素 P450 2B4 的能力。稳态酶动力学表明，连接体缩短 8-14 个氨基酸会抑制（63-99%）CYPOR 支持 CYP2B4 活性的能力，并显著增加 CYPOR 与 CYP2B4 的 K。此外，与野生型相比，当连接体缩短 8-14 个残基时，还原酶突变体降低了高铁细胞色素 P450 2B4 的还原速率（46-95%）。这些结果表明，具有最小长度为 8 个残基的连接体对于使还原酶的 FMN 结构域与 CYP2B4 相互作用以形成催化有效复合物是必需的。我们的研究提供了证据，表明 MBD-FMN 结构域连接体的长度是 CYPOR 支持 CYP 催化和 P450 酶药物代谢的能力的主要决定因素。前言：本文是为纪念 James R. Kincaid 博士而撰写的，他是 Freeborn Rwere 博士的博士导师，也是 Lucy Waskell 博士的长期合作者和朋友。James R. Kincaid 博士是化学专业的杰出教授，专门从事血红素蛋白的共振拉曼（rR）研究。他激励了 Rwere 博士（津巴布韦人）和另外三名津巴布韦人（Remigio Usai 博士、Daniel Kaluka 博士和 Munyaradzi E. Manyumwa 女士）使用激光记录球蛋白蛋白（肌红蛋白和血红蛋白）和细胞色素 P450 酶中血红素活性部位发生的细微变化。Rwere 博士感谢他为发展来自非洲的有才华的黑人科学家所做的贡献。
