Effects of heme pocket structure and mobility on cytochrome c stability

Unfolding thermodynamics of a thermophilic cytochrome c552 from Hydrogenobacter thermophilus (Ht cyt c552) and its mesophilic homologue from Pseudomonas aeruginosa (Pa cyt c551) as well as two heme pocket point mutants (Ht-Q64N and Pa-N64Q) are characterized by determination of protein stability curves (plots of unfolding free energy, DeltaG, vs T). These proteins show revealing differences in heme pocket hydrogen bonding and mobility. It previously has been shown that Asn64 in Pa cyt c551 and in Ht-Q64N interacts with the heme axial Met to fix it in a single conformation [Wen, X., and Bren, K. L. (2005) Biochemistry 44, 5225-5233]. In Ht cyt c552 and Pa-N64Q, Gln64 does not interact with the axial Met; in these variants the axial Met samples more than one conformation [Wen, X., and Bren, K. L. (2005) Inorg. Chem. 44, 8587-8593]. Here it is demonstrated that, relative to wild type, Pa-N64Q displays enhanced stability with an increase in unfolding free energy (DeltaDeltaG) of 7.1 kJ/mol and an increase in denaturation temperature (DeltaTm) of 8 degrees C. Correspondingly, Ht-Q64N is less stable than Ht cyt c552, with a DeltaDeltaG of -10 kJ/mol and a DeltaTm of -10 degrees C. Analysis of unfolding thermodynamics indicates that the net changes in stability resulting from the position 64 mutations are primarily attributable to entropic factors. For Pa-N64Q (Ht-Q64N) it is proposed that the favorable release (unfavorable burial) of residue 64 is the dominant factor impacting stability. The mobility of the axial Met also is proposed to contribute. These results provide a specific illustration of how amino acid side chain mobility and burial or release contribute to protein 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).     

Heme attachment motif mobility tunes cytochrome c redox potential

Hydrogen exchange (HX) rates and midpoint potentials (Em) of variants of cytochrome c from Pseudomonas aeruginosa (Pa cyt c551) and Hydrogenobacter thermophilus (Ht cyt c552) have been characterized in an effort to develop an understanding of the impact of properties of the Cys-X-X-Cys-His pentapeptide c-heme attachment (CXXCH) motif on heme redox potential. Despite structural conservation of the CXXCH motif, Ht cyt c552 exhibits a low level of protection from HX for amide protons within this motif relative to Pa cyt c551. Site-directed mutants have been prepared to determine the structural basis for and functional implications of these variations on HX behavior. The double mutant Ht-M13V/K22M displays suppressed HX within the CXXCH motif as well as a decreased Em (by 81 mV), whereas the corresponding double mutant of Pa cyt c551 (V13M/M22K) exhibits enhanced HX within the CXXCH pentapeptide and a modest increase in Em (by 30 mV). The changes in Em correlate with changes in axial His chemical shifts in the ferric proteins reflecting the extent of histidinate character. Thus, the mobility of the CXXCH pentapeptide is found to impact the His-Fe(III) interaction and therefore the heme redox potential.     

Effects of axial methionine coordination on the in-plane asymmetry of the heme electronic structure of cytochrome<Emphasis Type="Italic"> c</Emphasis>

The paramagnetic susceptibility () tensors of the oxidized forms of thermophile Hydrogenobacter thermophilus cytochrome c₅₅₂ (Ht cyt c₅₅₂) and a quintuple mutant (F7A/V13 M/F34Y/E43Y/V78I; qm) of mesophile Pseudomonas aeruginosa cytochrome c₅₅₁ (Pa cyt c₅₅₁) have been determined on the basis of the redox-dependent ¹H NMR shift changes of the main-chain NH and CH proton resonances of non-coordinated amino acid residues and the NMR structures of the reduced forms of the corresponding proteins (J. Hasegawa, T. Yoshida, T. Yamazaki, Y. Sambongi, Y. Yu, Y. Igarashi, T. Kodama, K. Yamazaki, Y. Kyogoku, Y. Kobayashi (1998) Biochemistry 37:9641–9649; J. Hasegawa, S. Uchiyama, Y. Tanimoto, M. Mizutani, Y. Kobayashi, Y. Sambongi,Y. Igarashi (2000) J Biol Chem 275:37824–37828). From the tensors determined, we obtained the contact shifts for heme methyl proton resonances, which provided the heme electronic structures of the oxidized forms of Ht cyt c₅₅₂ and qm. We also characterized the heme electronic structure of the cyanide adducts of the proteins, where the axial Met was replaced by an exogenous cyanide ion, through the analysis of ¹H NMR spectra. The results indicated that the heme electronic structures of both the proteins in their oxidized forms with axial His and Met coordination are largely different to each other, while those in their cyanide adducts are similar to each other. These results demonstrated that the orientation of the axial Met sulfur lone pair, with respect to heme, predominantly contributes to the spin delocalization into the porphyrin- system of heme in the oxidized proteins with axial His and Met coordination.Electronic Supplementary Material Supplementary material is available in the online version of this article at Abbreviations COSY correlation spectroscopy - DQF-COSY double quantum filtered COSY - TOCSY total correlation spectroscopy - NOE nuclear Overhauser effect - NOESY nuclear Overhauser effect correlated spectroscopy - Cyt c cytochrome c - Pa cyt c₅₅₁ Pseudomonas aeruginosa cytochrome c₅₅₁ - Ht cyt c₅₅₂ Hydrogenobacter thermophilus cytochrome c₅₅₂ - _obs observed shift - _para paramagnetic shift - _dia diamagnetic shift - _con contact shift - _pc pseudo-contact shift     

Domain-Swapped Dimer of Pseudomonas aeruginosa Cytochrome c 551: Structural Insights into Domain Swapping of Cytochrome c Family Proteins

Cytochrome c (cyt c) family proteins, such as horse cyt c, Pseudomonas aeruginosa cytochrome c ₅₅₁ (PA cyt c ₅₅₁), and Hydrogenobacter thermophilus cytochrome c ₅₅₂ (HT cyt c ₅₅₂), have been used as model proteins to study the relationship between the protein structure and folding process. We have shown in the past that horse cyt c forms oligomers by domain swapping its C-terminal helix, perturbing the Met–heme coordination significantly compared to the monomer. HT cyt c ₅₅₂ forms dimers by domain swapping the region containing the N-terminal α-helix and heme, where the heme axial His and Met ligands belong to different protomers. Herein, we show that PA cyt c ₅₅₁ also forms domain-swapped dimers by swapping the region containing the N-terminal α-helix and heme. The secondary structures of the M61A mutant of PA cyt c ₅₅₁ were perturbed slightly and its oligomer formation ability decreased compared to that of the wild-type protein, showing that the stability of the protein secondary structures is important for domain swapping. The hinge loop of domain swapping for cyt c family proteins corresponded to the unstable region specified by hydrogen exchange NMR measurements for the monomer, although the swapping region differed among proteins. These results show that the unstable loop region has a tendency to become a hinge loop in domain-swapped proteins.     

Protein surface charge effect on 3D domain swapping in cells for c-type cytochromes

Many c-type cytochromes (cyts) can form domain-swapped oligomers. The positively charged Hydrogenobacter thermophilus (HT) cytochrome (cyt) c₅₅₂ forms domain-swapped oligomers during expression in the Escherichia coli (E. coli) expression system, but the factors influencing the oligomerization remain unrevealed. Here, we found that the dimer of the negatively charged Shewanella violacea (SV) cyt c₅ exhibits a domain-swapped structure, in which the N-terminal helix is exchanged between protomers, similar to the structures of the HT cyt c₅₅₂ and Pseudomonas aeruginosa (PA) cyt c₅₅₁ domain-swapped dimers. Positively charged horse cyt c and HT cyt c₅₅₂ domain swapped during expression in E. coli, whereas negatively charged PA cyt c₅₅₁ and SV cyt c₅ did not. Oligomers were formed during expression in E. coli for HT cyt c₅₅₂ attached to either a co- or post-translational signal peptide for transportation through the cytoplasm membrane, but not for PA cyt c₅₅₁ attached to either signal peptide. HT cyt c₅₅₂ formed oligomers in E. coli in the presence and absence of rare codons. More oligomers were obtained from the in vitro folding of horse cyt c and HT cyt c₅₅₂ by the addition of negatively charged liposomes during folding, whereas the amount of oligomers for the in vitro folding of PA cyt c₅₅₁ and SV cyt c₅ did not change significantly by the addition. These results indicate that the protein surface charge affects the oligomerization of c-type cyts in cells; positively charged c-type cyts assemble on a negatively charged membrane, inducing formation of domain-swapped oligomers during folding.     

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.     

Membrane tetraheme cytochrome c(m552) of the ammonia-oxidizing nitrosomonas europaea: a ubiquinone reductase

Cytochrome c(m552) (cyt c(m552)) from the ammonia-oxidizing Nitrosomonas europaea is encoded by the cycB gene, which is preceded in a gene cluster by three genes encoding proteins involved in the oxidation of hydroxylamine: hao, hydroxylamine oxidoreductase; orf2, a putative membrane protein; cycA, cyt c(554). By amino acid sequence alignment of the core tetraheme domain, cyt c(m552) belongs to the NapC/TorC family of tetra- or pentaheme cytochrome c species involved in electron transport from membrane quinols to a variety of periplasmic electron shuttles leading to terminal reductases. However, cyt c(m552) is thought to reduce quinone with electrons originating from HAO. In this work, the tetrahemic 27 kDa cyt c(m552) from N. europaea was purified after extraction from membranes using Triton X-100 with subsequent exchange into n-dodecyl beta-d-maltoside. The cytochrome had a propensity to form strong SDS-resistant dimers likely mediated by a conserved GXXXG motif present in the putative transmembrane segment. Optical spectra of the ferric protein contained a broad ligand-metal charge transfer band at approximately 625 nm indicative of a high-spin heme. Mossbauer spectroscopy of the reduced (57)Fe-enriched protein revealed the presence of high-spin and low-spin hemes in a 1:3 ratio. Multimode EPR spectroscopy of the native state showed signals from an electronically interacting high-spin/low-spin pair of hemes. Upon partial reduction, a typical high-spin heme EPR signal was observed. No EPR signals were observed from the other two low-spin hemes, indicating an electronic interaction between these hemes as well. UV-vis absorption data indicate that CO (ferrous enzyme) or CN(-) (ferric or ferrous enzyme) bound to more than one and possibly all hemes. Other anionic ligands did not bind. The four ferrous hemes of the cytochrome were rapidly oxidized in the presence of oxygen. Comparative modeling, based on the crystal structure and conserved residues of the homologous NrfH protein from Desulfovibrio of cyt c(m552), predicted some structural elements, including a Met-ligated high-spin heme in a quinone-binding pocket, and likely axial ligands to all four hemes.     

Solution 1H NMR study of the active site structure for the double mutant H64Q/V68F cyanide complex from mouse neuroglobin

Solution (1)H NMR spectroscopy has been carried out to investigate the molecular and electronic structures of the active site in H64Q/V68F double mutant mouse neuroglobin in the cyanomet form. Two heme orientations resulting from a 180 degrees rotation about the alpha-gamma-meso axis were observed with a population ratio about 1:1, and the clearly distinguished B isomer was used to perform the study. Based on the analysis of the dipolar shifts and paramagnetic relaxation constants, the distal Gln(64)(E7) side chain is obtained to adopt an orientation that may produce hydrogen bond between the N(epsilon)H(1) and the Fe-bound cyanide. The side chain of Phe(68)(E11) is oriented out of the heme pocket just like that in triple mutant of cyanide complex of sperm whale myoglobin. A 15 degrees rotation of the imidazole ring in axial His(96) is observed, which is close to the varphi angle determined from the crystal structure of NgbCO. The quantitative determinations of the orientation and anisotropies of the paramagnetic susceptibility tensor reveal that cyanide is tilted by 8 degrees from the heme normal which allows for contact to the Gln(64)(E7) N(epsilon)H(1). The E7 and E11 residues appear to control the direction and the extent of tilt of the bound ligand. Furthermore, the tilt of the ligand has no obvious influence on the heme heterogeneity of cyanide ligation for isomer A/B of the wild type and mutant protein, indicating that factors other than steric effects, such as polarity of heme pocket, impacts on ligand binding affinity.     

Implication for using heme methyl hyperfine shifts as indicators of heme seating as related to stereoselectivity in the catabolism of heme by heme oxygenase: in-plane heme versus axial his rotation

The triple mutant of the solubilized, 265-residue construct of human heme oxygenase, K18E/E29K/R183E-hHO, has been shown to redirect the exclusive alpha-regioselectivity of wild-type hHO to primarily beta,delta-selectivity in the cleavage of heme (Wang, J., Evans, J. P., Ogura, H., La Mar, G. N., and Ortiz de Montellano, P. R. (2006) Biochemistry 45, 61-73). The 1H NMR hyperfine shift pattern for the substrate and axial His CbetaH's and the substrate-protein contacts of the cyanide-inhibited protohemin and 2,4-dimethyldeuterohemin complexes of the triple mutant have been analyzed in detail and compared to data for the WT complex. It is shown that protein contacts for the major solution isomers for both substrates in the mutant dictate approximately 90 degrees in-plane clockwise rotation relative to that in the WT. The conventional interpretation of the pattern of substrate methyl hyperfine shifts, however, indicates substrate rotations of only approximately 50 degrees . This paradox is resolved by demonstrating that the axial His25 imidazole ring also rotates counterclockwise with respect to the protein matrix in the mutant relative to that in the WT. The axial His25 CbetaH hyperfine shifts are shown to serve as independent probes of the imidazole plane orientation relative to the protein matrix. The analysis indicates that the pattern of heme methyl hyperfine shifts cannot be used alone to determine the in-plane orientation of the substrate as it relates to the stereospecificity of heme cleavage, without explicit consideration of the orientation of the axial His imidazole plane relative to the protein matrix.     

Cloning,heterologous expression,and characterization of recombinant class II cytochromes c from Rhodopseudomonas palustris

The cytochrome (cyt) c', cyt c(556), and cyt c(2) genes from Rhodopseudomonas palustris have been cloned; recombinant cyt c' and cyt c(556) have been expressed, purified, and characterized. Unlike mitochondrial cyt c, these two proteins are structurally similar to cyt b(562), in which the heme is embedded in a four-helix bundle. The hemes in both recombinant proteins form covalent thioether links to two Cys residues. UV/vis spectra of the Fe(II) and Fe(III) states of the recombinant cyts are identical with those of the corresponding native proteins. Equilibrium unfolding measurements in guanidine hydrochloride solutions confirm that native Fe(II)-cyt c(556) is more stable than the corresponding state of Fe(III)-cyt c(556) (DeltaDeltaG(f)(o) =22 kJ/mol).     

A new spatial structure for the axial methionine observed in cytochrome c5 from Pseudomonas mendocina. Correlations with the electronic structure of heme c

Cytochrome c5 from Pseudomonas mendocina has been isolated and the coordination geometry at the heme iron was investigated by 1H nuclear magnetic resonance and circular dichroism spectroscopy. Individual assignments were obtained for heme c and the axial ligands. From studies of nuclear Overhauser enhancements the axial histidine imidazole ring orientation relative to the heme group was found to coincide with that of other c-type cytochromes. In contrast, a new structure was observed for the axial methionine. This includes S chirality at the iron-bound sulfur atom, but compared to cytochromes c-551 from Pseudomonads and Rhodopseudomonas gelatinosa, which also contain S-chiral methionine, the spatial arrangement of the gamma- and beta-methylene groups and the alpha carbon of methionine is markedly different. Analysis of the electron spin density distribution in ferricytochrome c5 in the light of this new coordination geometry provides additional support for the hypothesis that the electronic structure of heme c is primarily governed by the orientation of the sp3 lone-pair orbital of the axial sulfur atom with respect to the heme plane.     

A model for the misfolded bis-His intermediate of cytochrome c: the 1-56 N-fragment

We have characterized the ferric and ferrous forms of the heme-containing (1-56 residues) N-fragment of horse heart cytochrome c (cyt c) at different pH values and low ionic strength by UV-visible absorption and resonance Raman (RR) scattering. The results are compared with native cyt c in the same experimental conditions as this may provide a deeper insight into the cyt c unfolding-folding process. Folding of cyt c leads to a state having the heme iron coordinated to a histidine (His18) and a methionine (Met80) as axial ligands. At neutral pH the N-fragment (which lacks Met80) shows absorption and RR spectra that are consistent with the presence of a bis-His low spin heme, like several non-native forms of the parental protein. In particular, the optical spectra are identical to those of cyt c in the presence of a high concentration of denaturants; this renders the N-fragment a suitable model to study the heme pocket microenvironment of the misfolded (His-His) intermediate formed during folding of cyt c. Acid pH affects the ligation state in both cyt c and the N-fragment. Data obtained as a function of pH allow a correlation between the structural properties in the heme pocket of the N-fragment and those of non-native forms of cyt c. The results underline that the (57-104 residues) segment under native-like conditions imparts structural stability to the protein by impeding solvent access into the heme pocket.     

An LHRH agonist inhibits FSH-induced cyclic AMP accumulation and steroidogenesis in porcine granulosa cells in culture

The heme iron coordination in horse cytochrome c and cytochrome c-551 from $\text{Pseudomonas aeruginosa}$ was investigated with ¹H NMR and CD spectroscopy. Truncated driven nuclear Overhauser enhancement (TOE) studies of the reduced proteins showed different chirality for the attachment of the axial methionine in the two species. For the oxidized proteins the different chirality was manifested in different CD properties of the 695 nm absorption band. Since additional NMR data indicated nearly identical coordination of the axial histidine in the two species it is suggested that the previously reported different electronic heme structures in the oxidized proteins are a consequence of the different mode of binding of the axial methionine.     

Electron nuclear double resonance and electron paramagnetic resonance study on the structure of the NO-ligated heme alpha 3 in cytochrome c oxidase

The techniques of EPR and electron nuclear double resonance (ENDOR) were used to probe structure and electronic distribution at the nitric oxide (NO)-ligated heme alpha 3 in the nitrosylferrocytochrome alpha 3 moiety of fully reduced cytochrome c oxidase. Hyperfine and quadrupole couplings to NO (in both 15NO and 14NO forms), to histidine nitrogens, and to protons near the heme site were obtained. Parallel studies were also performed on NO-ligated myoglobin and model NO-heme-imidazole systems. The major findings and interpretations on nitrosylferrocytochrome alpha 3 were: 1) compared to other NO-heme-imidazole systems, the nitrosylferrocytochrome alpha3 gave better resolution of EPR and ENDOR signals; 2) at the maximal g value (gx = 2.09), particularly well resolved NO nitrogen hyperfine and quadrupole couplings and mesoproton hyperfine couplings were seen. These hyperfine and quadrupole couplings gave information on the electronic distribution on the NO, on the orientation of the g tensor with respect to the heme, and possibly on the orientation of the FeNO plane; 3) a combination of experimental EPR-ENDOR results and EPR spectral simulations evidenced a rotation of the NO hyperfine tensor with respect to the electronic g tensor; this implied a bent Fe-NO bond; 4) ENDOR showed a unique proton not seen in the other NO heme systems studied. The magnitude of this proton's hyperfine coupling was consistent with this proton being part of a nearby protein side chain that perturbs an axial ligand like NO or O2.     

Conformation of the c552:aa3 electron transfer complex in Paracoccus denitrificans studied by EPR on oriented samples

The EPR spectral parameters of aa(3) oxidase and cyt c(552) from Paracoccus denitrificans were studied in purified oxidase and enriched cyt c(552). The orientation of the g-tensors of hemes a and c(552) were determined on partially ordered membranes, enriched cyt c(552) and a c(552):aa(3) subcomplex. The known correlation of g-tensor to molecular axes in histidine/methionine ligated hemes permits us to position cyt c(552) with respect to the parent membrane. Taken together with previous data on the interaction surface between aa(3) oxidase and cyt c(552), these results allow us to arrive at a single conformation for the c(552):aa(3) electron transfer complex.     

Individual 1H-NMR assignments for the heme groups and the axially bound amino acids and determination of the coordination geometry at the heme iron in a mixture of two isocytochromes c-551 from Rhodopseudomonas gelatinosa

This paper describes chemical and physicochemical studies of two small isocytochromes c-551 (approx. 9000 dalton) from Rhodopseudomonas gelatinosa. In spite of numerous amino acid substitutions in the N-terminal half of the sequence the two isoproteins could not be separated by the procedures used, presumably because they have identical size, charge and isoelectric points. Individual assignments of the 1H-NMR lines of heme c and the axial ligands to the heme iron were therefore obtained by nuclear Overhauser enhancement measurements and saturation transfer experiments in a mixed solution of the two isocytochromes c-551. The conformation of the coordination sphere was investigated by additional 1H-NMR and circular dichroism studies. For both isoproteins the electronic structure of the heme and the chirality of the methionine attachment to the iron were found to coincide with those in Pseudomonas cytochromes c-551, i.e., S chirality was observed for the axial methionine. The Rps. gelatinosa cytochromes c-551 thus differ from mammalian, yeast, Euglena gracilis and Rhodospirillum rubrum cytochromes c, which all have R chirality at the axial methionine and concomitantly a characteristically different electronic heme structure. This is the first observation of S chirality of the axially bound methionine in a species outside the Pseudomonas family. The redox potentials of the two isocytochromes c-551 of Rps. gelatinosa differ by approx. 120 mV, and there is no cross-exchange of electrons between the two species. The two isoproteins could thus function in two different, parallel electron-transfer chains or at two different locations in a single transfer sequence.     

Structure–function relationship of reduced cytochrome c probed by complete solution structure determination in 30% acetonitrile/water solution

The complete solution structure of ferrocytochrome c in 30% acetonitrile/70% water has been determined using high-field 1D and 2D (1)H NMR methods and deposited in the Protein Data Bank with codes 1LC1 and 1LC2. This is the first time a complete solution protein structure has been determined for a protein in nonaqueous media. Ferrocyt c retains a native protein secondary structure (five alpha-helices and two omega loops) in 30% acetonitrile. H18 and M80 residues are the axial heme ligands, as in aqueous solution. Residues believed to be axial heme ligands in the alkaline-like conformers of ferricyt c, specifically H33 and K72, are positioned close to the heme iron. The orientations of both heme propionates are markedly different in 30% acetonitrile/70% water. Comparative structural analysis of reduced cyt c in 30% acetonitrile/70% water solution with cyt c in different environments has given new insight into the cyt c folding mechanism, the electron transfer pathway, and cell apoptosis.     

Substitution of the sixth axial ligand of Rhodobacter capsulatus cytochrome c1 heme yields novel cytochrome c1 variants with unusual properties.

The cytochrome (cyt) c1 heme of the ubihydroquinone:cytochrome c oxidoreductase (bc1 complex) is covalently attached to two cysteine residues of the cyt c1 polypeptide chain via two thioether bonds, and the fifth and sixth axial ligands of its iron atom are histidine (H) and methionine (M), respectively. The latter residue is M183 in Rhodobacter capsulatus cyt c1, and previous mutagenesis studies revealed its critical role for the physicochemical properties of cyt c1 [Gray, K. A., Davidson, E., and Daldal, F. (1992) Biochemistry 31, 11864-11873]. In the homologous chloroplast b6f complex, the sixth axial ligand is provided by the amino group of the amino terminal tyrosine residue. To further pursue our investigation on the role played by the sixth axial ligand in heme-protein interactions, novel cyt c1 variants with histidine-lysine (K) and histidine-histidine axial coordination were sought. Using a R. capsulatus genetic system, the cyt c1 mutants M183K and M183H were constructed by site-directed mutagenesis, and chromatophore membranes as well as purified bc1 complexes obtained from these mutants were characterized in detail. The studies revealed that these mutants incorporated the heme group into the mature cyt c1 polypeptides, but yielded nonfunctional bc1 complexes with unusual spectroscopic and thermodynamic properties, including shifted optical absorption maxima (lambdamax) and decreased redox midpoint potential values (Em7). The availability and future detailed studies of these stable cyt c1 mutants should contribute to our understanding of how different factors influence the physicochemical and folding properties of membrane-bound c-type cytochromes in general.     

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.