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Fig. 4. Modulation by nitric oxide of fluorescence resonance energy transfer (FRET) in an MT-green fluorescent protein chimera. Top: Schematic of MT chimera, in which cDNA for hMTIIa is flanked by enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP) on the N and C termini. Under control conditions, MT is associated with seven zinc molecules and is in a relative folded conformation. Bottom: After exposure to EDTA (and NaCl) or medium that was gassed with NO to reach a final concentration of 80 ¬°iM, MT is predicted to lose zinc molecules and unfold. The conformational change is monitored by exciting the cells with a source at 440 nm and continuously monitoring the emission ratio of acceptor (535 nm) to donor (480 nm). SPAEC, sheep pulmonary artery endothelial cells. [The authors acknowledge Dr. Linda L. Pearce (University of Pittsburgh) for providing Fig. 4.]

liberation of free NO from nitrosothiol and its subsequent specific detection with the fluorogenic reagent 4,5-diaminofluorescein to produce a highly fluorescent tri-azole derivative.30 Interestingly enough, this approach reveals that only a small fraction (between 2 and 3%) of the total modified thiols in MT could be accounted for by S-NO. This suggests that the remainder of these modified MT cysteines underwent further oxidation after the initial nitrosylation event. A simple estimation of the disulfide portion among the oxidized cysteines can be performed with standard disulfide reducing reagents, such as DTT. Under the specific conditions outlined in Fig. 3 exposure of HL-60 cells to NO resulted in approximately 30% of DTT-recoverable cysteines in MT. The remaining 70% of the modified SH groups are presumably represented by higher S-oxides that were insensitive to DTT reduction. This is not surprising, given that the NO exposure was performed in aerobically incubated cell suspensions. It remains to be clarified whether this relatively high level of nonregenerable cysteines in MT is typical in vivo.

Summary

In summary, the redox conversions of MT cysteines are likely to be the principal mechanisms for regulation of metal binding and release by this protein. Oxidative and/or nitrosative challenges can serve to promote metal ion release from MT to render their delivery to specific target proteins. It is tempting to consider the potential roles of MTs as redox sensors because of their high sensitivity to cysteine modification, as well as their potential to amplify signals by releasing multiple metal ions. In other words, MTs may act early in a biological signaling cascade that triggers metal-dependent biochemical and cellular responses. Alternatively, uncontrolled release of metals by excessive oxidative stress may contribute to metal toxicity. Because oxidative and nitrosative signaling is ubiquitous within cells, the physiological function of MT demands that efficient recycling of modified cysteines be operative. Little is known regarding the potential mechanisms for the regeneration of MT after oxidative/nitrosative modification, but they may involve endogenous dithiols, such as thioredoxin, and pharmacologically relevant dithiols, such as dihydrolipoate.

Acknowledgments

This work was funded in part by an EPA STAR Grant (R827151) and by the NIH (HL-32154).

30 H. Kojima, N. Nakatsubo, K. Kikuchi, S. Kawahara, Y. Kirino, H. Nagoshi, Y. Hirata, and T. Nagano, Anal. Chem. 70, 2446 (1998).

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