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Compressing the free energy range of substructure stabilities in iso-1-cytochrome c.

ABSTRACT: Evolutionary conservation of substructure architecture between yeast iso-1-cytochrome c and the well-characterized horse cytochrome c is studied with limited proteolysis, the alkaline conformational transition and global unfolding with guanidine-HCl. Mass spectral analysis of limited proteolysis cleavage products for iso-1-cytochrome c show that its least stable substructure is the same as horse cytochrome c. The limited proteolysis data yield a free energy of 3.8 +/- 0.4 kcal mol(-1) to unfold the least stable substructure compared with 5.05 +/- 0.30 kcal mol(-1) for global unfolding of iso-1-cytochrome c. Thus, substructure stabilities of iso-1-cytochrome c span only approximately 1.2 kcal mol(-1) compared with approximately 8 kcal mol(-1) for horse cytochrome c. Consistent with the less cooperative folding thus expected for the horse protein, the guanidine-HCl m-values are approximately 3 kcal mol(-1)M(-1) versus approximately 4.5 kcal mol(-1)M(-1) for horse versus yeast cytochrome c. The tight free energy spacing of the yeast cytochrome c substructures suggests that its folding has more branch points than for horse cytochrome c. Studies on a variant of iso-1-cytochrome c with an H26N mutation indicate that the least and most stable substructures unfold sequentially and the two least stable substructures unfold independently as for horse cytochrome c. Thus, important aspects of the substructure architecture of horse cytochrome c, albeit compressed energetically, are preserved evolutionally in yeast iso-1-cytochrome c.

SUBMITTER: Duncan MG

PROVIDER: S-EPMC2774426 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Compressing the free energy range of substructure stabilities in iso-1-cytochrome c.

Duncan Michael G MG Williams Michael D MD Bowler Bruce E BE

Protein science : a publication of the Protein Society 20090601 6

Evolutionary conservation of substructure architecture between yeast iso-1-cytochrome c and the well-characterized horse cytochrome c is studied with limited proteolysis, the alkaline conformational transition and global unfolding with guanidine-HCl. Mass spectral analysis of limited proteolysis cleavage products for iso-1-cytochrome c show that its least stable substructure is the same as horse cytochrome c. The limited proteolysis data yield a free energy of 3.8 +/- 0.4 kcal mol(-1) to unfold ...[more]

PMID: 19472325

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Project description:BackgroundAcetogenic bacteria are able to use CO2 as terminal electron acceptor of an anaerobic respiration, thereby producing acetate with electrons coming from H2. Due to this feature, acetogens came into focus as platforms to produce biocommodities from waste gases such as H2+CO2 and/or CO. A prerequisite for metabolic engineering is a detailed understanding of the mechanisms of ATP synthesis and electron-transfer reactions to ensure redox homeostasis. Acetogenesis involves the reduction of CO2 to acetate via soluble enzymes and is coupled to energy conservation by a chemiosmotic mechanism. The membrane-bound module, acting as an ion pump, was of special interest for decades and recently, an Rnf complex was shown to couple electron flow from reduced ferredoxin to NAD+ with the export of Na+ in Acetobacterium woodii. However, not all acetogens have rnf genes in their genome. In order to gain further insights into energy conservation of non-Rnf-containing, thermophilic acetogens, we sequenced the genome of Thermoanaerobacter kivui.ResultsThe genome of Thermoanaerobacter kivui comprises 2.9 Mbp with a G+C content of 35% and 2,378 protein encoding orfs. Neither autotrophic growth nor acetate formation from H2+CO2 was dependent on Na+ and acetate formation was inhibited by a protonophore, indicating that H+ is used as coupling ion for primary bioenergetics. This is consistent with the finding that the c subunit of the F1FO ATP synthase does not have the conserved Na+ binding motif. A search for potential H+-translocating, membrane-bound protein complexes revealed genes potentially encoding two different proton-reducing, energy-conserving hydrogenases (Ech).ConclusionsThe thermophilic acetogen T. kivui does not use Na+ but H+ for chemiosmotic ATP synthesis. It does not contain cytochromes and the electrochemical proton gradient is most likely established by an energy-conserving hydrogenase (Ech). Its thermophilic nature and the efficient conversion of H2+CO2 make T. kivui an interesting acetogen to be used for the production of biocommodities in industrial micobiology. Furthermore, our experimental data as well as the increasing number of sequenced genomes of acetogenic bacteria supported the new classification of acetogens into two groups: Rnf- and Ech-containing acetogens.

Dataset Information

Compressing the free energy range of substructure stabilities in iso-1-cytochrome c.

Publications

Compressing the free energy range of substructure stabilities in iso-1-cytochrome c.

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