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dc.contributor.advisorGroves, John T
dc.contributor.authorImran, Syed Muhammad Saad
dc.contributor.otherChemistry Department
dc.description.abstractIron (Fe) is an essential element across the domains of life. Proteins bind Fe and significantly influence its reactivity. The two proteins discussed in this dissertation incorporate Fe in the form of an iron-sulfur (FeS) cluster or as the heme (Fe-protoporphyrin IX) cofactor. Part I of this dissertation relates to BchL, a FeS protein from Rhodobacter sphaeroides. BchL is part of a transient enzyme complex called dark-operative protochlorophyllide oxidoreductase. BchL-mediated electron transfer to the complex enables the formation of chlorophyllide in photosynthetic bacteria. A crystal structure of nucleotide-free BchL was solved, revealing previously unobserved N-terminal residues. These residues ‘cap’ the FeS cluster, providing a barrier to complex formation and electron transfer without nucleotides. Mutagenesis, sequence alignments, and spectroscopic experiments confirm a regulatory role of the N-terminus of BchL. Part II of the dissertation pertains to human indoleamine-2,3-dioxygenase 1 (IDO1), a heme enzyme that oxidizes the amino acid L-Tryptophan (L-Trp). IDO1 upregulation increases L-Trp oxidation, which enables immune escape in healthy and cancerous tissues, making IDO1 an important target for immune modulation. Catalysis by IDO1 proceeds via a transient ferryl (Fe(IV)=O) intermediate. A novel method of generating ferryl IDO1 was utilized, which revealed a high affinity interaction between L-Trp and ferryl IDO1. This off-cycle, stabilizing interaction was found to significantly contribute to the substrate inhibition of IDO1 and was relieved by physiologically relevant catechols. Stable mimics of reactive intermediates are useful for mechanistic and structural investigations. A molybdenyl (Mo(V)≡O) porphyrin was found to be a general ferryl heme mimic, capable of maintaining a permanent metal-oxo bond in heme proteins. Its incorporation in IDO1 and myoglobin was validated by several spectroscopic techniques. A vanadyl (V(IV)≡O) porphyrin was also explored but not found to be a general ferryl heme mimic. Cobalt protoporphyrin IX-substituted IDO1 (CoIDO1) was used to probe the heme-oxygen adduct formed during IDO1 catalysis. Modification of recombinant IDO1 expression yielded Co(III)IDO1. Co(II)IDO1 and oxygen reversibly formed an adduct which was stabilized by L-Trp, and incapable of turning over, yielding a ternary complex mimic. This work highlights how enzymes with both native and non-native cofactors can be mechanistically informative.
dc.publisherPrinceton, NJ : Princeton University
dc.subjectBiophysical techniques
dc.subjectEnzyme inhibition
dc.subjectEnzyme kinetics
dc.subjectHeme proteins
dc.subjectStructural biology
dc.titleInvestigations Into the Regulation and Mechanisms of Iron Proteins Involved in Chlorophyll Biosynthesis and Immune Modulation
dc.typeAcademic dissertations (Ph.D.)
Appears in Collections:Chemistry

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