Washington University in St. Louis Washington University Open Scholarship

Washington University Spring 2017 Senior Honors Thesis Abstracts

Spring 2017

Analysis of Human HCCS-Mediated Cytochrome c Biogenesis

Jennifer Hsu Washington University in St. Louis

Follow this and additional works at: https://openscholarship.wustl.edu/wushta_spr2017

Recommended Citation Hsu, Jennifer, "Analysis of Human HCCS-Mediated Cytochrome c Biogenesis" (2017). Spring 2017. 51. https://openscholarship.wustl.edu/wushta_spr2017/51

This Abstract for College of Arts & Sciences is brought to you for free and open access by the Washington University Senior Honors Thesis Abstracts at Washington University Open Scholarship. It has been accepted for inclusion in Spring 2017 by an authorized administrator of Washington University Open Scholarship. For more information, please contact [email protected]. Biology

Analysis of Human HCCS-Mediated Cytochrome c Biogenesis Jennifer Hsu

Mentors: Robert Kranz and Shalon Ledbetter

Cytochrome c (cyt c), found in the of mitochondria, is an essential component of cellular processes such as energy production and apoptosis. It acts as an electron carrier, and thus, defects in cyt c or its production, impair respiration. Holocyt c synthase (HCCS), a membrane-associated , is responsible for mediating the attachment of to cyt c. The stability and function of cyt c relies on attachment of heme via two thioether linkages at conserved cysteine residues

(Cys 15 XxxXxxCys 18 His 19 ). We characterized HCCS-mediated synthesis of several cyt c variants containing in and around the conserved motif to understand specific mechanisms of cyt c biosynthesis. Based on previous data from the Kranz lab regarding an enhanced cyt c product release feature of HCCS E159A, we successfully produced several cyt c variants in quantities suitable for biochemical characterization. My first study suggests that heme attachment preferentially begins at Cys18. We also show that mutations at Cys18 result in a cyt c species modified by an oxygen adduct, explaining the absence of Cys18 cyt c variants in nature. For my second related study, we show that residues outside the cyt c heme attachment motif and in the N-terminal alpha helix-1 position Cys15 of cyt c. We provide evidence that deletion of Met13 in alpha helix-1 displaces Cys15 away from heme 2-vinyl, thus preventing Cys15 thioether formation. In support of this conclusion, we show that further mutations to shift the positioning of Cys15 restore the two thioether conformation. My third study explores in vitro reconstitution of System III cyt c biogenesis to identify some optimal conditions for HCCS activity and required cyt c domains for HCCS recognition and attachment. My studies have contributed to understanding the mechanism of HCCS, an enzyme required by most eukaryotes.

36 Spring 2017 WUSHTA