Hiroyasu Tachikawa

Surface-enhanced resonance Raman spectroscopic characterization of the protein native structure.

Surface-enhanced resonance Raman scattering (SERRS) spectra of biological species are often different from their resonance Raman (RR) spectra. A home-designed Raman flow system is used to determine the factors that contribute to the difference between the SERRS and RR of met-myoglobin (metMb). The results indicate that both the degree of protein-nanoparticles interaction and the laser irradiation contribute to the structural changes and are responsible for the observed differences between the SERRS and RR spectra of metMb. The prolonged adsorption of the protein molecules on the nanoparticle surface, which is the condition normally used for the conventional SERRS experiments, disturbs the heme pocket structure and facilitates the charge transfer process and the photoinduced transformation of proteins. The disruption of the heme pocket results in the loss of the distal water molecule, and the resulting SERRS spectrum of metMb shows a 5-coordinated high-spin heme. The flow system, when operated at a moderately high flow rate, can basically eliminate the factors that disturb the protein structure while maintaining a high enhancement factor. The SERRS spectrum obtained from a 1 x 10 (-7) M metMb solution using this flow system is basically identical to the RR spectrum of a 5 x 10 (-4) M metMb solution. Therefore, the Raman flow system reported here should be useful for characterizing the protein-nanoparticles interaction and the native structure of proteins using SERRS spectroscopy.

Functional importance of tyrosine 294 and the catalytic selectivity for the bis-Fe(IV) state of MauG revealed by replacement of this axial heme ligand with histidine .

The diheme enzyme MauG catalyzes the posttranslational modification of a precursor protein of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. It catalyzes three sequential two-electron oxidation reactions which proceed through a high-valent bis-Fe(IV) redox state. Tyr294, the unusual distal axial ligand of one c-type heme, was mutated to His, and the crystal structure of Y294H MauG in complex with preMADH reveals that this heme now has His-His axial ligation. Y294H MauG is able to interact with preMADH and participate in interprotein electron transfer, but it is unable to catalyze the TTQ biosynthesis reactions that require the bis-Fe(IV) state. This mutation affects not only the redox properties of the six-coordinate heme but also the redox and CO-binding properties of the five-coordinate heme, despite the 21 Å separation of the heme iron centers. This highlights the communication between the hemes which in wild-type MauG behave as a single diheme unit. Spectroscopic data suggest that Y294H MauG can stabilize a high-valent redox state equivalent to Fe(V), but it appears to be an Fe(IV)═O/π radical at the five-coordinate heme rather than the bis-Fe(IV) state. This compound I-like intermediate does not catalyze TTQ biosynthesis, demonstrating that the bis-Fe(IV) state, which is stabilized by Tyr294, is specifically required for this reaction. The TTQ biosynthetic reactions catalyzed by wild-type MauG do not occur via direct contact with the Fe(IV)═O heme but via long-range electron transfer through the six-coordinate heme. Thus, a critical feature of the bis-Fe(IV) species may be that it shortens the electron transfer distance from preMADH to a high-valent heme iron.

Electrochemical determination of thiols at single-wall carbon nanotubes and PQQ modified electrodes.

The electrocatalytic oxidation of thiols has been observed at a glassy carbon (GC) electrode coated with a single-wall carbon nanotube (SWNT) film. Fourteen thiols including L-cysteine (CySH) and glutathione were tested using the SWNT/GC electrode, and the cyclic voltammetry (CV) showed that each thiol was oxidized at much less positive potential than those at other electrodes such as bare GC and diamond electrodes. The SWNT/GC electrode was also modified with pyrroloquinoline quinone (PQQ) which showed a further improvement of the catalytic behavior of the SWNT/GC electrode: e.g. the oxidation peak current of CySH was observed at 0.27 V vs. Ag/AgCl in pH 7.5 phosphate buffer. The amperometic responses at these electrodes showed a linear relationship with the substrate concentration in a 10(-6)-10(-3) M range and 10(-6)-10(-7) M detection limits for several thiols including CySH, L-homocysteine, N-acetyl-L-cysteine, L-penicillamine and glutathione. These electrodes show a response time of 2-3 s and storage stabilities over 3 weeks. A PQQ/SWNT/GC electrode has been successfully applied for the assay of both L-cysteine and N-acetyl-L-cysteine in the dietary supplement.

Initiation Reactions of Lipid Peroxidation: A Raman Spectroscopic and Quantum-Mechanical Study

Near-infrared (NIR) Raman spectroscopy has been used to observe the spectrum changes of linolenic acid during the iron-dependent lipid peroxidation reactions. The intensity of several bands including those at 726, 977, 1264, 1658, and 3014 cm−1 decreased after lipid peroxidation. Other notable changes after lipid peroxidation include the appearance of a couple of new bands at 1165 and 1638 cm−1. The Raman spectrum change was suppressed by an antioxidant, 21-aminosteroid. Harmonic ab initio vibrational analysis was performed for two model structures: one for the pure linolenic acid and another for a peroxidation product. The calculations by the Hartree–Fock (HF) method and a density functional theory (DFT) indicate that a new band which appeared at 1638 cm−1 after lipid peroxidation is due to the rearrangement of skeletal double bonds.

CHAPTER 15 – Electrochemical sensors based on carbon nanotubes

Publisher Summary This chapter focuses on the electrochemical sensors based on carbon nanotubes (CNT). The various advantages CNTs for electrochemical sensor applications include their small size with large surface area, high sensitivity, fast response time, enhanced electron transfer, easy protein immobilization with retention of activity, and alleviation of surface fouling effects. There are various methods used for forming CNT-modified electrodes which can be used for detecting analytes. Among the various types of transducers based on CNTs, the CNT-composite electrode was the first CNT electrode tested in 1996, and is still widely used with different composite materials such as conducting polymers, nanoparticles and sol–gel. These days, more applications are based on the layer-by-layer fabrication techniques for CNT-modified electrodes. This technique clearly provides thinner and more isolated CNTs compared with other methods such as CNT-composite and CNT coated electrodes in which CNTs are in the form of big bundles. This method should help biomolecules such as enzymes and DNA to interact more effectively with CNTs than other methods, and sensors based on this technique are expected to be more sensitive. Important biosensors such as glucose sensors have been developed by using this technique, and further development of other sensors based on the layer-by-layer technique is expected. Vertically aligned CNT-modified electrodes are based on a more elaborate technique, and microscopic images are used to characterize the integrity of this type of electrode. This technique has been applied in the immobilization of enzymes and DNA, and the sensors based on this technique have shown a lower detection limit than those based on other methods.