Graduation Year


Date of Thesis Acceptance

Fall 12-9-2016

Major Department or Program



Timothy Machonkin


Non-heme iron enzymes are a wide class of proteins that complete a myriad of highly specified and biologically important functions. The mechanistic details of their specificity and reactivity have yet to be fully elucidated. PcpA, a ring-cleaving dioxgenase (RCDO) with a high degree of specificity for ortho-halogenated hydroquinone substrates, is an atypical enzyme of the class that offers insights into the importance of active site coordination geometry, substrate character, and proton control in the activity of RCDOs. A purification procedure was developed for the wild-type PcpA expressed in and purified from E. coli along with crystallization procedures for the purpose of structural studies of the wild-type enzyme. Synthetic models mimicking common RCDO active site moieties and substrate types were also developed. Models incorporating phenol and hydroquinone motifs in addition to a facially coordinating triad of N and O ligands were created and complexed with divalent first row transition metals. N-methylamino linked iminodiacetic acid (IDA) and histadine (His) moieties were joined with a variety of substituted phenol and hydroquinone substrates, isolated, and characterized. 2-(N-methylamino-IDA)-hydroquinone (L1), 4-methoxy-2,6-di(Nmethylamino- IDA)-phenol (L2), 6-tertbutyl-4-methoxy-2-(N-methylamino-IDA)- phenol (L4), and 4-methoxy-2-(N-methylamino-His)-phenol (L3) were synthesized and characterized via 1H-NMR. Ni(II), Co(II) and Fe(II) metallations were attempted for all ligands and the resulting complexes characterized in part via paramagnetic 1HNMR, Mössbauer spectroscopy, UV-Vis spectroscopy, cyclic voltammetry, and single crystal x-ray diffractometry. Crystal structures of L2-Co(II)2 and L4-Co(II) confirmed initial estimates of coordination geometry of IDA-based ligands. Structures of L4 complexes, vi showing distinct 1:1 binding:non-binding repeating modes in the crystal, suggest that ortho-tertbutyl substituted linked substrates may be unsuitable for further development due to steric crowding around the coordination site compared to other complexes. Spectroscopic titrations of L4-M(II) complexes confirmed significant pH dependence for chelation and substrate protonation state in aqueous phase complexes of this type. The dinucleating ligand, L2, was found to result from the preferential overreaction of 2,6-unsubstituted phenol in a Mannich-type reaction suggesting that future ligand syntheses would benefit from phenol 6-position substitutions to ensure only monosubstitution at the 2-position resulting in mononucleating products.

Page Count


Subject Headings

Enzymes -- Synthesis -- Research -- Testing, Ion exchange chromatography -- Purification -- Development -- PcpA protein, Mannich reaction -- Evolution -- Derivatives, Ligands (Biochemistry) -- Production standards -- Study and teaching, Whitman College 2017 -- Dissertation collection -- Chemistry Department

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Document Type

Whitman Community Accessible Thesis

Available for download on Sunday, December 09, 2018



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