Chad E. Immoos

Conformationally Gated Electrochemical Gene Detection

The synthesis and characterization of a 26‐base DNA hairpin containing both a redox‐active reporter (ferrocene) and terminal thiol functionality for electrochemical gene detection is described. This electrochemical DNA sensor exploits electron‐transfer dynamics that alter as a consequence of a large structural rearrangement (hairpin‐to‐duplex) induced by hybridization of the target DNA sequence. Melting temperature and circular dichroism studies confirm that the 26‐mer DNA forms a hairpin structure in the absence of target DNA. The loop region of the DNA hairpin is shown to form a stable duplex in the presence of complementary single‐stranded DNA. Atomic force microscopy and ellipsometry experiments of immobilized self‐assembled DNA monolayers suggest that hybridization with complementary DNA affords a conformational change that alters the electrochemical response.

A Novel Heme and Peroxide-Dependent Tryptophan-Tyrosine Cross-Link in a Mutant of Cytochrome c Peroxidase

The crystal structure of a cytochrome c peroxidase mutant where the distal catalytic His52 is converted to Tyr reveals that the tyrosine side-chain forms a covalent bond with the indole ring nitrogen atom of Trp51. We hypothesize that this novel bond results from peroxide activation by the heme iron followed by oxidation of Trp51 and Tyr52. This hypothesis has been tested by incorporation of a redox-inactive Zn-protoporphyrin into the protein, and the resulting crystal structure shows the absence of a Trp51-Tyr52 cross-link. Instead, the Tyr52 side-chain orients away from the heme active-site pocket, which requires a substantial rearrangement of residues 72-80 and 134-144. Additional experiments where heme-containing crystals of the mutant were treated with peroxide support our hypothesis that this novel Trp-Tyr cross-link is a peroxide-dependent process mediated by the heme iron.

Electrochemistry of the Cu_A domain of Thermus thermophilus cytochrome ba _3

Abstract The electrochemistry of a water-soluble fragment from the CuA domain of Thermus thermophilus cytochrome ba3 has been investigated. At 25  °C, CuA exhibits a reversible reduction at a pyridine-4-aldehydesemicarbazone-modified gold electrode (0.1 M Tris, pH 8) with E° = 0.24 V vs NHE. Thermodynamic parameters for the [Cu(Cys)2Cu]+/0 electrode reaction were determined by variable-temperature electrochemistry (ΔS°rc = –5.4(12) eu, ΔS° = –21.0(12) eu, ΔH° = –11.9(4) kcal/mol;ΔG° = –5.6 (11) kcal/mol). The relatively small reaction entropy is consistent with a low reorganization energy for [Cu(Cys)2Cu]+/0 electron transfer. An irreversible oxidation of [Cu(Cys)2Cu]+ at 1 V vs NHE confirms that the CuII:CuII state of CuA is significantly destabilized relative to the CuII state of analogous blue-copper proteins.

Electron transfer in the ruthenated heme domain of cytochrome P450BM‐3

Two mutants of the heme domain of Bacillus megaterium cytochrome P450BM-3 (BMP) have been modified by covalent attachment of a photoactive Ru(bpy)3 complex at the surface-exposed cysteine residues, 62 and 387. The laser-flash/quench technique was used to study Ru1+ Fe3+ electron transfer (ET) within the Ru-BMP complexes. There was no reduction of the ferric heme by Ru1+ in Ru-62-BMP. In Ru-387-BMP, ET from Ru1+ to Fe3+ occurred with the rate constant of 4.6 × 105 s−1 and 2.5 × 106 s−1 in the absence and presence of substrate, respectively. The study demonstrates the importance of through-bond pathways for electron flow to the heme iron of P450 and that the Gln387–Cys400 peptide is a potential ET root in P450BM-3.

Supramolecular assemblies of DNA with neutral nucleoside amphiphiles

A neutral uridine-based amphiphile is described which condenses plasmid DNA. AFM studies show that the three distinct structural components of the amphiphile (i.e, nucleobase, alkyl chains, and poly(ethylene glycol)) are required for the formation of DNA-amphiphile supramolecular assemblies on a mica surface.

Mesopone Cytochrome c Peroxidase: Functional Model of Heme Oxygenated Oxidases

The effect of heme ring oxygenation on enzyme structure and function has been examined in a reconstituted cytochrome c peroxidase. Oxochlorin derivatives were formed by OsO(4) treatment of mesoporphyrin followed by acid-catalyzed pinacol rearrangement. The northern oxochlorin isomers were isolated by chromatography, and the regio-isomers assignments determined by 2D COSY and NOE 1H NMR. The major isomer, 4-mesoporphyrinone (Mp), was metallated with FeCl(2) and reconstituted into cytochrome c peroxidase (CcP) forming a hybrid green protein, MpCcP. The heme-altered enzyme has 99% wild-type peroxidase activity with cytochrome c. EPR spectroscopy of MpCcP intermediate compound I verifies the formation of the Trp(191) radical similar to wild-type CcP in the reaction cycle. Peroxidase activity with small molecules is varied: guaiacol turnover increases approximately five-fold while that with ferrocyanide is approximately 85% of native. The electron-withdrawing oxo-substitutents on the cofactor cause a approximately 60-mV increase in Fe(III)/Fe(II) reduction potential. The present investigation represents the first structural characterization of an oxochlorin protein with X-ray intensity data collected to 1.70 A. Although a mixture of R- and S-mesopone isomers of the FeMP cofactor was used during heme incorporation into the apo-protein, only the S-isomer is found in the crystallized protein.

Characterization of Reagent Pencils for Deposition of Reagents onto Paper-Based Microfluidic Devices

Reagent pencils allow for solvent-free deposition of reagents onto paper-based microfluidic devices. The pencils are portable, easy to use, extend the shelf-life of reagents, and offer a platform for customizing diagnostic devices at the point of care. In this work, reagent pencils were characterized by measuring the wear resistance of pencil cores made from polyethylene glycols (PEGs) with different molecular weights and incorporating various concentrations of three different reagents using a standard pin abrasion test, as well as by measuring the efficiency of reagent delivery from the pencils to the test zones of paper-based microfluidic devices using absorption spectroscopy and digital image colorimetry. The molecular weight of the PEG, concentration of the reagent, and the molecular weight of the reagent were all found to have an inverse correlation with the wear of the pencil cores, but the amount of reagent delivered to the test zone of a device correlated most strongly with the concentration of the reagent in the pencil core. Up to 49% of the total reagent deposited on a device with a pencil was released into the test zone, compared to 58% for reagents deposited from a solution. The results suggest that reagent pencils can be prepared for a variety of reagents using PEGs with molecular weights in the range of 2000 to 6000 g/mol.