Namdoo Moon

Regulation of the soxRS Oxidative Stress Regulon REVERSIBLE OXIDATION OF THE Fe-S CENTERS OF SoxR IN VIVO

SoxR protein, a transcriptional activator of the soxRS (superoxide response) regulon of Escherichia coli, contains autooxidizable [2Fe-2S] centers that are presumed to serve as redox sensors. In vitro transcription experiments previously demonstrated that only the oxidized form is active. Reduced SoxR was detected in overproducing strains by EPR spectroscopy of suspensions of intact cells. Oxidized Fe-S centers were determined by lysing the cells and treating them with the reducing agent sodium dithionite prior to EPR measurements. In uninduced cells, 90% of the SoxR was in the reduced form. Treatment with the redox cycling agents phenazine methosulfate or plumbagin was accompanied by reversible oxidation of the Fe-S centers. Mutant SoxR derivatives that were constitutively activated existed constitutively in an oxidized state. The results indicate the presence of a cellular pathway for countering the autooxidation of SoxR and confirm the hypothesis that induction of the regulon is mediated by a shift in the redox equilibrium of SoxR rather than by assembly of its Fe-S clusters.

Identification and functional requirement of Cu(I) and its ligands within coagulation factor VIII.

Coagulation factor VIII (FVIII) is a heterodimer consisting of a light chain of 80 kDa (domains A3-C1-C2) in a metal ion-dependent association with a 220-kDa heavy chain (domains A1-A2-B). The nature of the metal ion-dependent association between the heavy and light chains was investigated using atomic absorption spectroscopy, electron paramagnetic resonance spectroscopy (EPR), and site-directed mutagenesis and expression of the FVIII cDNA. Whereas copper ion was not detected in intact recombinant FVIII, EDTA dissociation of the chains yielded an EPR signal consistent with 1 mol of Cu(I)/mol of active protein, supporting the hypothesis that a single molecule of reduced copper ion is buried within intact FVIII and is released and oxidized upon treatment with EDTA. Cu(I), and not Cu(II), was able to reconstitute FVIII activity from dissociated chains, demonstrating a requirement for Cu(I) in FVIII function. Three potential copper ion binding sites exist within FVIII: one type-2 site and two type-1 sites. The importance of these potential copper ion ligands was tested by studying the effect of site-directed mutants. Of the two histidines that compose the type-2 binding site, the His-1957 → Ala mutant displayed secretion, light and heavy chain assembly, and activity similar to wild-type FVIII, while mutant His-99 → Ala was partially defective for secretion and had low levels of heavy and light chain association and activity. In contrast, FVIII having the mutation Cys-310 → Ser within the type-1 copper binding site in the A1 domain was inactive and partially defective for secretion from the cell, and the heavy and light chains of the secreted protein were not associated. Mutant Cys-2000 → Ser within the A3 domain displayed secretion, assembly, and activity similar to that for wild-type FVIII. These results support the hypothesis that Cu(I) is buried within the type-1 copper binding site within the A1 domain and is required for FVIII chain association and activity.

The high-potential flavin and heme of nitric oxide synthase are not magnetically linked: implications for electron transfer

BACKGROUND The homodimeric nitric oxide synthase (NOS) catalyzes conversion of L-arginine to L-citrulline and nitric oxide. Each subunit contains two flavins and one protoporphyrin IX heme. A key component of the reaction is the transfer of electrons from the flavins to the heme. The NOS gene encodes two domains linked by a short helix containing a calmodulin-recognition sequence. The reductase domain binds the flavin cofactors, while the oxygenase domain binds heme and L-arginine and additionally mediates the dimerization of the NOS subunits. We investigated the origin of the unusual magnetic properties (rapid-spin relaxation) of an air-stable free radical localized to a reductase domain flavin cofactor. RESULTS We characterized the air-stable flavin in wild-type NOS, both in the presence and absence of calcium and calmodulin, the imidazole-bound heme complex of wild-type NOS, the NOS Cys415-->Ala mutant, and the isolated reductase domain. All preparations of NOS had the same flavin electron-spin relaxation behavior. No half-field transitions or temperature-dependent changes in the linewidth of the radical spin signal were detected. CONCLUSIONS These data suggest that the observed relaxation enhancement of the NOS flavin radical is caused by the environment provided by the reductase domain. No magnetic interaction between the heme and flavin cofactors was detected, suggesting that the flavin and heme centers are probably separated by more than 15 A.

The effect of three Korean traditional medicines on the growth rate of cultured human keratinocytes

The effect of three different Korean Traditional Medicines (KTM) was studied on several functional parameters of adult human cells in culture. The cells were non-transformed strains of normal, skin epidermal cells (keratinocytes) from adult humans. Aqueous extracts of the herbal medicines were tested using two types of cell strains: one type was essential fatty acid deficient (EFAD) cells which grow rapidly in medium that was low in calcium and had no essential fatty acids; the second type was a cell strain grown in medium supplemented with essential fatty acid (EFA-supplemented). These cells had much slower, in vivo skin growth rates, and the fatty acid composition resembled that measured in epidermal biopsy tissue. The KTMs chosen for this study were tae-gang-hual-tang (for treating osteoarthritis), hual-ak-tang (for pain relief) and sip-zeon-tae-bo-tang (for fortifying immune systems). Because high proliferation rates usually correlate with skin inflammation and because many of the chemotactic agents mediating inflammatory response are modified fatty acids, this study focused on cell growth rate and membrane fatty acid composition as signals for the effects of the herbal medicines. By monitoring growth rate, these experiments measured both a stimulatory and a regulatory effect on the growth of keratinocytes. Some toxicity was seen at the highest doses of the KTMs. These effects were modeled mathematically, and the results showed varying effects on growth rate depending on dose and herbal recipe. The fitting parameters were discussed as they relate to biological function. The experimental design was also discussed and alternatives were suggested.

A high-sensitivity Mössbauer spectrometer facilitates the study of iron proteins at natural abundance

Abstract A Wide-Angle Mossbauer Spectrometer has been constructed, tested and shown to have approximately ten times the sensitivity of a standard Mossbauer spectrometer. The most innovative feature of the spectrometer is the eighty degree, conical acceptance geometry of its gamma-ray detector, which is constructed of 77 argon gas proportional counters with their associated charge-sensitive preamplifiers and single channel analyzers. The spectrometer has demonstrated the following features: (1) it has approximately 100 times the countrate of most existing Mossbauer spectrometers; (2) velocities reproduce to an accuracy of ±0.005 mm/s over periods of several weeks; (3) the temperatures of the source and the sample are regulated with an accuracy of ±0.1°C in the range of 4.2 to 300 K. By obtaining a high quality Mossbauer spectrum of un-enriched 0.3 mM K 3 Fe(CN) 6 , we have shown that the study of high molecular weight natural abundance iron proteins can be greatly facilitated by this spectrometer.