Debasish Kuila

Resonance Raman studies of Rieske-type proteins

Resonance Raman (RR) spectra are reported for the [2Fe-2S] Rieske protein from Thermus thermophilus (TRP) and phthalate dioxygenase from Pseudomonas cepacia (PDO) as a function of pH and excitation wavelength. Depolarization ratio measurements are presented for the RR spectra of spinach ferredoxin (SFD), TRP, and PDO at 74 K. By comparison with previously published RR spectra of SFD, we suggest reasonable assignments for the spectra of TRP and PDO. The spectra of PDO exhibit virtually no pH dependence, while significant changes are observed in TRP spectra upon raising the pH from 7.3 to 10.1. One band near 270 cm-1, which consists of components at 266 cm-1 and 274 cm-1, is attributed to Fe(III)-N(His) stretching motions. We suggest that these two components arise from conformers having a protonated-hydrogen-bonded imidazole (266 cm-1) and deprotonated-hydrogen-bonded imidazolate (274 cm-1) coordinated to the Fe/S cluster and that the relative populations of the two species are pH-dependent; a simple structural model is proposed to account for this behavior in the respiratory-type Rieske proteins. In addition, we have identified RR peaks associated with the bridging and terminal sulfur atoms of the Fe-S-N cluster. The RR excitation profiles of peaks associated with these atoms are indistinguishable from each other in TRP (pH 7.3) and PDO and differ greatly from those of [2Fe-2S] ferrodoxins. The profiles are bimodal with maxima near 490 nm and > approx. 550 nm. By contrast, bands associated with the Fe-N stretch show a somewhat different enhancement profile. Upon reduction, RR peaks assigned to Fe-N vibrations are no longer observed, with the resulting spectrum being remarkably similar to that reported for reduced adrenodoxin. This indicates that only modes associated with Fe-S bonds are observed and supports the idea that the reducing electron resides on the iron atom coordinated to the two histidine residues. Taken as a whole, the data are consistent with an St2FeSb2Fe[N(His)]t2 structure for the Rieske-type cluster.

Comparison of the spin-lattice relaxation properties of the two classes of (2Fe-2S) clusters in proteins

Two classes of [2Fe-2S] proteins have been defined according to the mean value gav of their g tensor components (Bertrand, P., Guigliarelli, B., Gayda, J.P., Beardwood, P. and Gibson, J.F. (1985) Biochim. Biophys. Acta 831, 261-266). To characterize their magnetic properties better, we have compared the spin-lattice relaxation behavior of typical proteins which belong to these two classes, namely Spirulina maxima and adrenal ferredoxin for the gav approximately 1.96 class, Thermus thermophilus Rieske protein and Pseudomonas putida benzene dioxygenase for the gav approximately 1.91 class. For all these proteins, the data support the existence of an efficient Orbach process in the highest temperature range, which allows the determination of the exchange coupling parameter, J. From the comparison of the J values obtained in each class, it is concluded that the structural factors which determine the value of the g tensor and the strength of the antiferromagnetic exchange interactions are different.

Polyacrylic acid (poly-A) as a chelant and dispersant

Polyacrylic acid was synthesized in water by persulfate-initiated polymerization (solution polymerization) of glacial acrylic acid in the absence of a chain-transfer agent. The final product is odorless and colorless. Chelation for calcium ions using a calcium electrode show that our poly(acrylic acid) has a higher chelation capacity than that of existing commercial poly(acrylic acids). A design of experiments was performed to optimize the synthesis conditions to obtain poly(acrylic acid) with a high maximum chelation value. These studies also helped us to gain insight into its high chelation capacity. The chelation capacity for calcium reaches its highest values when polymerization near isothermal conditions is done ∼ 95°C with an acrylic acid concentration of ≤21 wt % and an addition time >1 h. These conditions favor higher molecular weight poly(acrylic acid) with a polydispersity ∼ 4. The dispersion properties of our poly(acrylic acid) are similar to those of the commercial ones. This dual capability of chelation and dispersion is absent in commercial chelants such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), and their analogs. At pH > 7, chelation of calcium by our poly(acrylic acid) is much higher than that observed with EDTA. Characterization by NMR, Raman, FTIR, and molecular modeling are included in an attempt to understand structural features that can explain the higher chelation capacity of our atactic poly(acrylic acid).