A flexible linker of 8-amino acids between the membrane binding segment and the FMN domain of cytochrome P450 reductase is necessary for optimal activity

• Published in: Journal of inorganic biochemistry Vol. 259; p. 112667
• Main Authors: Rwere, Freeborn, Cartee, Naw May P., Yang, Yuting, Waskell, Lucy
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.10.2024
• Subjects: Animals; Aryl Hydrocarbon Hydroxylases - chemistry; Aryl Hydrocarbon Hydroxylases - genetics; Aryl Hydrocarbon Hydroxylases - metabolism; Benzphetamine; Cytochrome c; Cytochrome P450 2B4; Cytochrome P450 Family 2 - chemistry; Cytochrome P450 Family 2 - genetics; Cytochrome P450 Family 2 - metabolism; Cytochrome P450 reductase; Enzyme kinetics; Flavin Mononucleotide - chemistry; Flavin Mononucleotide - metabolism; Flexible linker; Humans; Kinetics; Mutagenesis, Site-Directed; NADPH-Ferrihemoprotein Reductase - chemistry; NADPH-Ferrihemoprotein Reductase - genetics; NADPH-Ferrihemoprotein Reductase - metabolism; Protein Domains; Cytochrome c; Benzphetamine; Flexible linker; Enzyme kinetics; Cytochrome P450 2B4; Cytochrome P450 reductase
• ISSN: 0162-0134; 1873-3344; 1873-3344
• DOI: 10.1016/j.jinorgbio.2024.112667

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. 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. The linker between the membrane binding domain (MBD) and FMN domain of cytochrome P450 reductase (CYPOR) help CYPOR to interact with cytochrome P450 2B4 (CYP2B4) to form a catalytically competent complex. Deleting the linker by 14-amino acids impairs the interaction between CYPOR and CYP2B4 to form a functional complex. Resultantly, the 14-amino acid deletion mutant is essentially inactive with CYP2B4. [Display omitted] •Reductase require a linker with at least 8 residues to form a catalytically competent complex with cytochrome P450 (CYP) enzymes.•Shortening the linker by 8–14 residues reduced the ability of reductase to support CYP2B4 activity.•Shortening the linker has no effect on the interaction of reductase with non-physiologic redox partners.•Electron transfer to ferric CYP2B4 was perturbed by deleting 8–14 residues in the linker.