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Thermophile Hydrogenobacter thermophilus cytochrome c552 (HT) is a stable protein, which has a denaturation temperature [T.sub.m)) of 109.8 and 129.7 degree Celsius for the oxidized and reduced forms, respectively. The removal of a single hydroxyl group from the hydrophobic core of HT, through the replacement of a Tyr by Phe, was found to result in further elevation of the [T.sub. m) value of the oxidized form by approximately 6 degree Celsius, the [T.sub. m) value of the reduced one remaining essentially unaltered.

Pseudomonas aeruginosa cytochrome c(sub 551) and a series of its mutants exhibiting various thermostabilities are studied by paramagnetic (super 1)H NMR and cyclic voltammetry to elucidate the molecular mechanics responsible for control of the redox potentials [E(super o')] of the proteins. The study revealed that the [E(super o')] value of the protein is regulated by two molecular mechanisms operating independently of each other.

Metal centers in metalloproteins involve multiple metal-ligand bonds. The release of metal ions from metalloproteins can have significant biological consequences, so understanding of the mechanisms by which metal ion dissociates has broad implications. By definition, the release of metal ions from metalloproteins involves the disruption of multiple metal-ligand bonds, and this process is often accompanied by unfolding of the protein. Detailed pathways for metal ion release from metalloproteins have been difficult to elucidate by classical ensemble techniques. Here, we combine single molecule force spectroscopy and protein engineering techniques to investigate the mechanical dissociation mechanism of iron from the active site of the simplest iron-sulfur protein, rubredoxin, at the single molecule level. Our results reveal that the mechanical rupture of this simplest iron center is stochastic and follows multiple, complex pathways that include concurrent rupture of multiple ferric-thiolate bonds as well as sequential rupture of ferric-thiolate bonds that lead to the formation of intermediate species. Our results uncover the surprising complexity of the rupture process of the seemingly simple iron center in rubredoxin and provide the first unambiguous experimental evidence concerning the detailed mechanism of mechanical disruption of a metal center in its native protein environment in aqueous solution. This study opens up a new avenue to investigating the rupture mechanism of metal centers in metalloproteins with unprecedented resolution by using single molecule force spectroscopy techniques. [ABSTRACT FROM AUTHOR]

It has long been recognized that hydrogen bonds formed by protein backbone amides with cysteinyl Sγ atoms play important roles in modulating the functional and structural properties of the iron-sulfur centers in proteins. Here we use single molecule atomic force microscopy, cyclic voltammetry, and protein engineering techniques to investigate directly how the strength of N-H…Sγ hydrogen bonds in the secondary coordination sphere affects the mechanical stability of Fe(III)-thiolate bonds of rubredoxin. Our results show that the mechanical stability of Fe(III)-thiolate bonds in rubredoxin correlates with the strength of N-H…Sγ hydrogen bonds as reflected by the midpoint reduction potential, providing direct evidence that N-H…Sγ hydrogen bonds play important roles in modulating the mechanical and kinetic properties of the Fe(III)-thiolate bonds of iron-sulfur proteins and corroborating the important roles of the protein environment in tuning the properties of metal-thiolate bonds. [ABSTRACT FROM AUTHOR]

The article presents a study which examines the correlation between protein stability and redox potential of homologous cytochromes c from Hydrogenobacter thermophilus, Hydrogenophilus thermoluteolus and Pseudomonas aeruginosa. It notes that the stability of the three cytochromes c was monitored by Soret absorption measurements in the concentrations of a denaturant and guanidine thiocyanate.

In order to elucidate the molecular mechanisms responsible for the apparent nonlinear behavior of the temperature dependence of the redox potential of Hydrogenobacter thermophilus cytochrome c552 [Takahashi, Y., Sasaki, H., Takayama, S. J., Mikami, S., Kawano, S., Mita, H., Sambongi, Y., and Yamamoto, Y. (2006) Biochemistry 45, 11005-11011], its heme active site structure has been characterized using variable-temperature and -pressure NMR techniques. The study revealed a temperature-dependent conformational transition between protein structures, which slightly differ in the conformation of the loop bearing the Fe-bound axial Met residue. The heme environment in the protein structure which arises at lower temperature was found to be more polar, as a result of the altered orientation of the loop with respect to the heme due to its conformational change, than that arising at higher temperature. The present study demonstrated the importance of the structural and dynamic properties of the polypeptide chain in close proximity to the heme for redox regulation of the protein. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa cytochrome C551 and a series of its mutants exhibiting various thermostabilities have been studied by paramagnetic ¹H NMR and cyclic voltammetry in an effort to elucidate the molecular mechanisms responsible for control of the redox potentials (E°') of the proteins. The study revealed that the E°' value of the protein is regulated by two molecular mechanisms operating independently of each other. One is based on the Fe-Met coordination bond strength in the protein, which is determined by the amino acid side chain packing in the protein, and the other on the pKa of the heme 17-propionic acid side chain, which is affected by the electrostatic environment. The former mechanism alters the magnitude of the E°' value throughout the entire pH range, and the latter regulates the pK values reflected by the pH profile of the E°' value. These findings provide novel insights into functional regulation of the protein, which could be utilized for tuning the E°' value of the protein by means of protein engineering. [ABSTRACT FROM AUTHOR]

The influence of a single amide group located in the heme active site of Pseudomonas aeruginosa cytochrome c551 on its redox potential (Eº′) has been characterized through amino acid substitution of Asn64 with Gln or Ala. The Eº′ variation of ca. 60 mV among the proteins is attributed mainly to the alteration of the thermodynamic stability of the oxidized protein by the dipole and hydrogen-bonding ability of the amide group. [ABSTRACT FROM AUTHOR]

The influence of a single amide group located in the heme active site of Pseudomonas aeruginosa cytochrome c551 on its redox potential (Eº′) has been characterized through amino acid substitution of Asn64 with Gln or Ala. The Eº′ variation of ca. 60 mV among the proteins is attributed mainly to the alteration of the thermodynamic stability of the oxidized protein by the dipole and hydrogen-bonding ability of the amide group. [ABSTRACT FROM AUTHOR]

Thermophile Hydrogenobacter thermophilus cytochrome c552 (HT) is a stable protein with denaturation temperatures (Tm) of 109.8 and 129.7 °C for the oxidized and reduced forms, respectively [Uchiyama, S., Ohshima, A., Nakamura, S., Hasegawa, J., Terui, N., Takayama, S. J., Yamamoto, Y., Sambongi, Y., and Kobayashi, Y. (2004) J. Am. Chem. Soc. 126, 14684–14685]. The removal of a single hydroxyl group from the hydrophobic core of HT, through the replacement of a Tyr by Phe, resulted in further elevation of the Tm value of the oxidized form by ∼6 °C, the Tm value of the reduced one remaining essentially unaltered. As a result, the redox potential of the mutant with higher stability in the oxidized form exhibited a negative shift of ∼20 mV relative to that of wild-type HT in an enthalpic manner. These findings indicated that the redox function of a protein can be enthalpically regulated through the stability of the oxidized form by altering the contextual stereochemical packing of hydrophobic residues in the protein interior using protein engineering. [ABSTRACT FROM AUTHOR]