The entatic state plays a key role in modulating the functional properties of metal sites, especially in proteins. In cytochrome (cyt) c, a hydrogen-bonding network contributes to stabilization Show more
The entatic state plays a key role in modulating the functional properties of metal sites, especially in proteins. In cytochrome (cyt) c, a hydrogen-bonding network contributes to stabilization of Met80 ligation to the heme iron, supporting the dual functions of this metalloprotein as an electron carrier in respiration and a peroxidase in apoptosis. We have prepared a cyt c variant in which both Thr49 and Thr78 within this network have been replaced with Val residues. Spectroscopic and electrochemical experiments suggest that the ferric form of the protein no longer has Met80 coordinating the heme iron, while the ferrous form preserves this interaction. Thermodynamic analyses demonstrate how perturbations at the periphery of the heme introduced by the mutations affect the stabilities of Met-, Lys-, and H2O-ligated conformers. The foldon structure enables the propagation of destabilization effects to the region implicated in the entatic control of the Met80 ligation. The extent of destabilization is similar for the ferric and ferrous Met-ligated conformers, but the ligation outcome differs because the global stability of the protein and stabilities of its foldons depend on the redox state of the heme iron. The stability of low-energy foldons could be tuned in other metalloproteins to engineer redox-linked switchable functions. Show less
The two roles of cytochrome c (cyt c), in oxidative phosphorylation and apoptosis, critically depend on redox properties of its heme iron center. The K79G mutant has served as a parent protein for a s Show more
The two roles of cytochrome c (cyt c), in oxidative phosphorylation and apoptosis, critically depend on redox properties of its heme iron center. The K79G mutant has served as a parent protein for a series of mutants of yeast iso-1 cyt c. The mutation preserves the Met80 coordination to the heme iron, as found in WT* (K72A/C102S), and many spectroscopic properties of K79G and WT* are indistinguishable. The K79G mutation does not alter the global stability, fold, rate of Met80 dissociation, or thermodynamics of the alkaline transition (p Ka) of the protein. However, the reduction potential of the heme iron decreases; further, the p KH of the trigger group and the rate of the Met-to-Lys ligand exchange associated with the alkaline transition decrease, suggesting changes in the environment of the heme. The rates of electron self-exchange and bimolecular electron transfer (ET) with positively charged inorganic complexes increase, as does the intrinsic peroxidase activity. Analysis of the reaction rates suggests that there is increased accessibility of the heme edge in K79G and supports the importance of the Lys79 site for bimolecular ET reactions of cyt c, including those with some of its native redox partners. Structural modeling rationalizes the observed effects to arise from changes in the volume of the heme pocket and solvent accessibility of the heme group. Kinetic and structural analyses of WT* characterize the properties of the heme crevice of this commonly employed reference variant. This study highlights the important role of Lys79 for defining functional redox properties of cyt c. Show less
Abstract TFIIH is a 10âsubunit complex that regulates RNA polymerase II (pol II) transcription but also serves other important biological roles. Although much remains unknown about TFIIH function in Show more
Abstract TFIIH is a 10âsubunit complex that regulates RNA polymerase II (pol II) transcription but also serves other important biological roles. Although much remains unknown about TFIIH function in eukaryotic cells, much progress has been made even in just the past few years, due in part to technological advances (e.g. cryoEM and single molecule methods) and the development of chemical inhibitors of TFIIH enzymes. This review focuses on the major cellular roles for TFIIH, with an emphasis on TFIIH function as a regulator of pol II transcription. We describe the structure of TFIIH and its roles in pol II initiation, promoterâproximal pausing, elongation, and termination. We also discuss cellular roles for TFIIH beyond transcription (e.g. DNA repair, cell cycle regulation) and summarize small molecule inhibitors of TFIIH and diseases associated with defects in TFIIH structure and function. Show less