Further enhancement of the thermostability of Hydrogenobacter thermophilus cytochrome c552

Thermophile Hydrogenobacter thermophilus cytochrome c(552) (HT) is a stable protein with denaturation temperatures (T(m)) of 109.8 and 129.7 degrees 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 T(m) value of the oxidized form by approximately 6 degrees C, the T(m) 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 approximately 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.     

An obligatory intermediate in the folding pathway of cytochrome c552 from Hydrogenobacter thermophilus

The folding mechanism of many proteins involves the population of partially organized structures en route to the native state. Identification and characterization of these intermediates is particularly difficult, as they are often only transiently populated and may play different mechanistic roles, being either on-pathway productive species or off-pathway kinetic traps. Following different spectroscopic probes, and employing state-of-the-art kinetic analysis, we present evidence that the folding mechanism of the thermostable cytochrome c552 from Hydrogenobacter thermophilus does involve the presence of an elusive, yet compact, on-pathway intermediate. Characterization of the folding mechanism of this cytochrome c is particularly interesting for the purpose of comparative folding studies, because H. thermophilus cytochrome c552 shares high sequence identity and structural homology with its homologue from the mesophilic bacterium Pseudomonas aeruginosa cytochrome c551, which refolds through a broad energy barrier without the accumulation of intermediates. Analysis of the folding kinetics and correlation with the three-dimensional structure add new evidence for the validity of a consensus folding mechanism in the cytochrome c family.     

Crystallization and preliminary X-ray diffraction study of cytochrome c552 from Hydrogenobacter thermophilus.

Cytochrome c552 from a thermophilic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus, exhibits remarkable thermostability. The oxidized cytochrome c552 has been crystallized in an ethanol/water mixture by means of the vapor diffusion method. The crystals belong to the orthorhombic system, space group P2(1)2(1)2, with unit cell dimensions of a = 93.4 A, b = 52.9 A, and c = 32.4 A. Most probably the asymmetric unit contains two molecules of cytochrome c552. The crystals diffract X-rays to better than 2.5 A resolution and are stable to X-ray irradiation.     

Local conformational transition of Hydrogenobacter thermophilus cytochrome c552 relevant to its redox potential

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.     

Thermostability of cytochrome c-552 from the thermophilic hydrogen-oxidizing bacterium Hydrogenobacter thermophilus

The denaturation of the c-type cytochrome of the thermophilic bacterium Hydrogenobacter thermophilus cytochrome c-552 by heat and guanidine hydrochloride was studied by measuring the change in circular dichroic spectra. The melting temperature (T1/2) of cytochrome c-552 in the presence of 1.5 M guanidine hydrochloride was 34 degrees C higher than that of the c-type cytochrome of Pseudomonas aeruginosa cytochrome c-551. Hydrogenobacter cytochrome c-552 is a much more stable protein than cytochrome c-551 of the mesophilic bacterium P. aeruginosa, even though their amino acid sequences are 56% identical and they have numerous other similarities. However, notwithstanding these similarities between the sequences of the cytochromes c-552 and c-551 that were compared, it is very likely that these differences in stability could be due to some heretofore undefined differences in their spatial structures. It has been suggested that alpha-helix structure and electrostatic interaction could be the source of the stable spatial structure of cytochrome c-552.     

NMR analysis shows that a b-type variant of Hydrogenobacter thermophilus cytochrome c552 retains its native structure

Conversion of Hydrogenobacter thermophilus cytochrome c(552) into a b-type cytochrome by mutagenesis of both heme-binding cysteines to alanines significantly reduces the stability of the protein (Tomlinson, E. J., and Ferguson, S. J. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 5156-5160). To understand the effects of this change on the structure and dynamics of the protein, hetero-nuclear (15)N-edited NMR techniques have been used to characterize this b-type variant. The backbone (15)N, (1)H(N), and (1)H(alpha), and (1)H(beta) resonances of the protein have been assigned. Analysis of (3)J(HN)alpha coupling constants, nuclear Overhauser enhancement intensities, and chemical shift index data demonstrates that the four alpha-helices present in the wild-type protein are retained in the b-type variant. Comparison of the chemical shifts for the b-type and wild-type proteins indicates that the tertiary structures of the two proteins are closely similar. Some subtle differences are, however, observed for residues in the N-terminal region and in the vicinity of the heme-binding pocket. Hydrogen exchange studies show that there are 25 backbone amide protons that exchange very slowly in the b-type variant and confirm that the fluctuations within the b-type protein are of a similar extent to those in the wild-type protein. These data demonstrate the notable retention of the native secondary structure and tertiary fold despite the absence of covalent linkages between the heme group and the protein.     

Five amino acid residues responsible for the high stability of Hydrogenobacter thermophilus cytochrome c552: reciprocal mutation analysis

Five amino acid residues responsible for extreme stability have been identified in cytochrome c(552) (HT c(552)) from a thermophilic bacterium, Hydrogenobacter thermophilus. The five residues, which are spatially distributed in three regions of HT c(552), were replaced with the corresponding residues in the homologous but less stable cytochrome c(551) (PA c(551)) from Pseudomonas aeruginosa. The quintuple HT c(552) variant (A7F/M13V/Y34F/Y43E/I78V) showed the same stability against guanidine hydrochloride denaturation as that of PA c(551), suggesting that the five residues in HT c(552) necessarily and sufficiently contribute to the overall stability. In the three HT c(552) variants carrying mutations in each of the three regions, the Y34F/Y43E mutations resulted in the greatest destabilization, by -13.3 kJ mol(-1), followed by A7F/M13V (-3.3 kJ mol(-1)) and then I78V (-1.5 kJ mol(-1)). The order of destabilization in HT c(552) was the same as that of stabilization in PA c(551) with reverse mutations such as F34Y/E43Y, F7A/V13M, and V78I (13.4, 10.3, and 0.3 kJ mol(-1), respectively). The results of guanidine hydrochloride denaturation were consistent with those of thermal denaturation for the same variants. The present study established a method for reciprocal mutation analysis. The effects of side-chain contacts were experimentally evaluated by swapping the residues between the two homologous proteins that differ in stability. A comparative study of the two proteins was a useful tool for assessing the amino acid contribution to the overall stability.     

Characterization of Hydrogenobacter thermophilus cytochromes c 552 expressed in the cytoplasm and periplasm of Escherichia coli

Hydrogenobacter thermophilus cytochrome c(552) ( Ht cyt c(552)) is a small monoheme protein in the cytochrome c(551) family. Ht cyt c(552) is unique because it is hypothesized to undergo spontaneous cytoplasmic maturation (covalent heme attachment) when expressed in Escherichia coli. This is in contrast to the usual maturation route for bacterial cytochromes c that occurs in the cellular periplasm, where maturation factors direct heme attachment. Here, the expression of Ht cyts c(552) in the periplasm as well as the cytoplasm of E. coli is reported. The products are characterized by absorption, circular dichroism, and NMR spectroscopy as well as mass spectrometry, proteolysis, and denaturation studies. The periplasmic product's properties are found to be indistinguishable from those reported for protein isolated from Ht cells, while the major cytoplasmic product exhibits structural anomalies in the region of the N-terminal helix. These anomalies are shown to result from the retention of the N-terminal methionine in the cytoplasmic product, and not from heme attachment errors. The (1)H NMR chemical shifts of the heme methyls of the oxidized ( S=1/2) expression products display a unique pattern not previously reported for a cytochrome c with histidine-methionine axial ligation, although they are consistent with native-like heme ligation. These results support the hypothesis that proper heme attachment can occur spontaneously in the E. coli cytoplasm for Ht cyt c(552).     

Stabilization of Pseudomonas aeruginosa cytochrome c(551) by systematic amino acid substitutions based on the structure of thermophilic Hydrogenobacter thermophilus cytochrome c(552)

A heterologous overexpression system for mesophilic Pseudomonas aeruginosa holocytochrome c(551) (PA c(551)) was established using Escherichia coli as a host organism. Amino acid residues were systematically substituted in three regions of PA c(551) with the corresponding residues from thermophilic Hydrogenobacter thermophilus cytochrome c(552) (HT c(552)), which has similar main chain folding to PA c(551), but is more stable to heat. Thermodynamic properties of PA c(551) with one of three single mutations (Phe-7 to Ala, Phe-34 to Tyr, or Val-78 to Ile) showed that these mutants had increased thermostability compared with that of the wild-type. Ala-7 and Ile-78 may contribute to the thermostability by tighter hydrophobic packing, which is indicated by the three dimensional structure comparison of PA c(551) with HT c(552). In the Phe-34 to Tyr mutant, the hydroxyl group of the Tyr residue and the guanidyl base of Arg-47 formed a hydrogen bond, which did not exist between the corresponding residues in HT c(552). We also found that stability of mutant proteins to denaturation by guanidine hydrochloride correlated with that against the thermal denaturation. These results and others described here suggest that significant stabilization of PA c(551) can be achieved through a few amino acid substitutions determined by molecular modeling with reference to the structure of HT c(552). The higher stability of HT c(552) may in part be attributed to some of these substitutions.     

Amino acid sequence of cytochrome c-552 from a thermophilic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus.

The complete amino acid sequence of cytochrome c-552 from an extremely thermophilic hydrogen bacterium, Hydrogenobacter thermophilus TK-6 (IAM 12695), was determined. It is a single polypeptide chain of 80 residues, and its molecular weight, including heme, was calculated to be 7,599. The sequence of cytochrome c-552 from H. thermophilus TK-6 closely resembles that of cytochromes c-551 from Pseudomonas species. Moreover, the tertiary structure of Hydrogenobacter cytochrome c-552 is suggested to be similar to that of cytochrome c-551 from Pseudomonas aeruginosa. The sequence similarity between Hydrogenobacter cytochrome c-552 and that of other bacteria physiologically related to H. thermophilus is not high.     

Enhancement of the thermostability of Hydrogenobacter thermophilus cytochrome c(552) through introduction of an extra methylene group into its hydrophobic protein interior

Careful scrutiny of the protein interior of Hydrogenobacter thermophilus cytochrome c(552) (HT) on the basis of its X-ray structure [Travaglini-Allocatelli, C., Gianni, S., Dubey, V. K., Borgia, A., Di Matteo, A., Bonivento, D., Cutruzzola, F., Bren, K. L., and Brunori, M. (2005) J. Biol. Chem. 280, 25729-25734] indicated that a void space, which is large enough to accommodate a methyl group, exists in the hydrophobic protein interior near the heme. We tried to reduce the void space through the replacement of a Val by Ile or Leu (Val/Ile or Val/Leu mutation), and then the structural and functional consequences of these two mutations were characterized in order to elucidate the relationship between the nature of the packing of hydrophobic residues and the functional properties of the protein. The study demonstrated striking differences in the structural and functional consequences between the two mutations. The Val/Ile mutation was found to cause further enhancement of the thermostability of the oxidized HT, as reflected in the increase of the denaturation temperature (T(m)) value by ～ 3 deg, whereas the thermostability of the reduced form was essentially unaffected. As a result, the redox potential (E(m)) of the Val/Ile mutant exhibited a negative shift of ～ 50 mV relative to that of the wild-type protein in an enthalpic manner, this being consistent with our previous finding that a protein with higher stability in its oxidized form exhibits a lower E(m) value [Terui, N., Tachiiri, N., Matsuo, H., Hasegawa, J., Uchiyama, S., Kobayashi, Y., Igarashi, Y., Sambongi, Y., and Yamamoto, Y. (2003) J. Am. Chem. Soc. 125, 13650-13651]. In contrast, the Val/Leu mutation led to a decrease in thermostability of both the redox forms of the protein, as reflected in the decreases of the T(m) values of the oxidized and reduced proteins by ～ 3 and ～ 5 deg, respectively, and the E(m) value of the Val/Leu mutant happened to be similar to that of the Val/Ile one. The E(m) value of the Val/Leu mutant could be reasonably interpreted in terms of the different effects of the mutation on the stabilities of the two different redox forms of the protein. Thus, the present study demonstrated that the stability of the protein is affected quite sensitively by the contextual stereochemical packing of hydrophobic residues in the protein interior and that the structural properties of the hydrophobic core in the protein interior are crucial for control of the redox function of the protein. These findings provide novel insights as to functional control of a protein, which could be utilized for tuning of the T(m) and E(m) values of the protein by means of protein engineering.     

Molecular dynamics simulations of Hydrogenobacter thermophilus cytochrome c552: comparisons of the wild-type protein, a b-type variant, and the apo state

Molecular dynamic simulations have been performed for wild-type Hydrogenobacter thermophilus cytochrome c(552), a b-type variant of the protein, and the apo state with the heme prosthetic group removed. In the b-type variant, Cys 10 and Cys 13 were mutated to alanine residues, and so the heme group was no longer covalently bound to the protein. Two 8-ns simulations have been performed for each system at 298 and 360 K. The simulations of the wild-type protein at 298 K show a very close agreement with experimental NMR data. A fluxional process involving the side chain of Met 59, which coordinates to the heme iron, is observed in accord with proposals from NMR studies. Overall, the structure and dynamical behavior of the protein during the simulations of the b-type variant is closely similar to that of the wild-type protein. However, side chains in the heme-binding site show larger fluctuations in the b-type variant simulation at 360 K. In addition, structural changes are seen for a number of residues close to the heme group, particularly Gly 22 and Ser 51. The simulations of the apo state show significant conformational changes for residues 50-59. These residues form a loop region, which packs over the heme group in the wild-type protein and hydrogen bonds to the heme propionate groups. In the absence of heme, in the apo state simulations, these residues form short but persistent regions of beta-sheet secondary structure. These could provide nucleation sites for the conversion to amyloid fibrils.     

Exploring the cytochrome c folding mechanism: cytochrome c552 from thermus thermophilus folds through an on-pathway intermediate

Understanding the role of partially folded intermediate states in the folding mechanism of a protein is a crucial yet very difficult problem. We exploited a kinetic approach to demonstrate that a transient intermediate of a thermostable member of the widely studied cytochrome c family (cytochrome c552 from Thermus thermophilus) is indeed on-pathway. This is the first clear indication of an obligatory intermediate in the folding mechanism of a cytochrome c. The fluorescence properties of this intermediate demonstrate that the relative position of the heme and of the only tryptophan residue cannot correspond to their native orientation. Based on an analysis of the three-dimensional structure of cytochrome c552, we propose an interpretation of the data which explains the residual fluorescence of the intermediate and is consistent with the established role played by some conserved interhelical interactions in the folding of other members of this family. A limited set of topologically conserved contacts may guide the folding of evolutionary distant cytochromes c through the same partially structured state, which, however, can play different kinetic roles, acting either as an intermediate or a transition state.     

In vitro studies on thioether bond formation between Hydrogenobacter thermophilus apocytochrome c(552) with metalloprotoporphyrin derivatives

Previously, in vitro formation of thioether bonds between Hydrogenobacter thermophilus apocytochrome c(552) and Fe-protoporphyrin IX has been demonstrated. Now we report studies on the reaction between the metalloderivatives Zn-, Co-, and Mn-protoporphyrin IX and the cysteine thiols of H. thermophilus apocytochrome c(552). All of these metalloporphyrins were capable of forming a "b-type cytochrome" state in which the hydrophobic prosthetic group is bound non-covalently. Zn(II)-protoporphyrin IX attached to the polypeptide covalently in the presence of either dithiothreitol or tri(2-carboxyethyl)phosphine to keep the thiol moieties reduced. These data show that the chemical nature of the thiol-reducing agent does not interfere with the thioether bond-forming mechanism. Mn-porphyrin could only react with the protein in the divalent state of the metal ion. Co-porphyrin did not react with the cysteine thiols of the apocytochrome in either oxidation state of the metal. In the absence of a metal (i.e. protoporphyrin IX itself), no reactivity toward apocytochrome is observed. These results have significant implications for the chemical requirements for thioether bond formation of heme vinyl groups to cysteine thiols and also have potential applications in de novo design of metalloproteins.     

NMR and CD conformational studies of the C-terminal 16-peptides of Pseudomonas aeruginosa c551 and Hydrogenobacter thermophilus c552 cytochromes.

The 16-amino acid sequences of the C-terminal helices of the homologous bacterial cytochromes c551 from Pseudomonas aeruginosa and C552 from Hydrogenobacter thermophilus were synthesized and their solution structure studied. Circular dichroism and NMR experiments in aqueous solution have shown the presence of alpha-helices and 3(10)-helices. The populations of helical structures in phosphate buffer, pH 3.5, 293 K, were 21% for c551 and 20% for c552, but increased to 56.7 and 48%, respectively, in 50% aqueous 2,2,2-trifluoroethanol. An isodichroic point was observed at 203 nm in CD spectra for the helix/coil transition in mixtures of water/2,2,2-trifluoroethanol. NMR spectra in phosphate buffer show the presence of both alpha- and 3(10)-helical structures. In water/2,2,2-trifluoroethanol (50:50) alpha-helices are predominant. CD temperature-dependency studies indicate that both peptides exhibit the same cooperativity for the transition in water/2,2,2-trifluoroethanol (50:50). The experimental data show that the amino acid substitutions do not favor heat resistance of the secondary structure of the c552 C-terminal helix at the local level. Instead, they optimize nonlocal contacts of the polypeptide chain, which stabilize the tertiary structure in the native protein.     

Amino acid sequence of cytochrome c-552 from Thermus thermophilus HB8

The complete amino acid sequence of thermophilic cytochrome c-552 from Thermus thermophilus HB8 is presented. The 131-residue sequence was derived by analysis of three cyanogen bromide fragments of the S-carboxymethylated apo-protein and their subpeptides. The sequence is homologous to c-type cytochromes, especially in the heme-binding region.