Date of Award

8-2026

Degree Name

Master of Science

Department

Biological Sciences

First Advisor

Todd Barkman, Ph.D.

Second Advisor

Frederick Stull, Ph.D.

Third Advisor

Benjamin Koestler, Ph.D.

Keywords

enzyme, flavin-dependent, HOSCN

Access Setting

Masters Thesis-Open Access

Abstract

Hypothiocyanous acid reductase (Har) is a flavin-dependent enzyme that is effective in protecting bacteria from hypothiocyanous acid. HOSCN is an antimicrobial oxidant that is excreted by the mammalian innate immune system during inflammation, targeting and attacking thiols in pathogens. To combat HOSCN, bacteria have developed defense systems, including hypothiocyanous acid reductase. The catalytic mechanism by which Har interacts with its substrates, HOSCN and NADH, is undetermined and requires exploration.

In this study, we characterized the reductive and oxidative half-reactions of E. coli Har (EcHar). We first investigated and characterized wild-type EcHar, showing that Har is extremely efficient in reacting with HOSCN (kobs > 1000 s-1) in the oxidative half-reaction and that the rate limiting step of the reductive half-reaction differs with NADH concentration. Secondly, this study investigated the roles of different active site amino acids in the reductive and oxidative half-reactions. Site directed mutagenesis of specific well-known and unique active site amino acids in Har gives insight on the specific roles that the amino acids play during catalysis. This study shows that the histidine/glutamate diad has an impactful role as an acid-base catalyst and that the unique lysine residues both have impactful roles in the oxidative and reductive half-reactions. The results further elucidate the catalytic mechanism of Har, which gives us insight on novel approaches to target Har during the immune response so that pathogenic bacteria may become more susceptible to HOSCN.

Comments

Todd Barkman, Ph.D., chair

Frederick Stull, Ph.D., co-chair

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Bacteriology Commons

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