Native Cyt Research Articles

A functional hybrid between the cytochrome bc1 complex and its physiological membrane-anchored electron acceptor cytochrome cy in Rhodobacter capsulatus

The membrane integral ubihydroquinone (QH 2): cytochrome (cyt) c oxidoreductase (or the cyt bc 1 complex) and its physiological electron acceptor, the membrane-anchored cytochrome c y (cyt c y), are discrete components of photosynthetic and respiratory electron transport chains of purple non-sulfur, facultative phototrophic bacteria of Rhodobacter species. In Rhodobacter capsulatus, it has been observed previously that, depending on the growth condition, absence of the cyt bc 1 complex is often correlated with a similar lack of cyt c y (Jenney, F. E., et al. (1994) Biochemistry 33, 2496–2502), as if these two membrane integral components form a non-transient larger structure. To probe whether such a structural super complex can exist in photosynthetic or respiratory membranes, we attempted to genetically fuse cyt c y to the cyt bc 1 complex. Here, we report successful production, and initial characterization, of a functional cyt bc 1- c y fusion complex that supports photosynthetic growth of an appropriate R. capsulatus mutant strain. The three-subunit cyt bc 1- c y fusion complex has an unprecedented bis-heme cyt c 1- c y subunit instead of the native mono-heme cyt c 1, is efficiently matured and assembled, and can sustain cyclic electron transfer in situ. The remarkable ability of R. capsulatus cells to produce a cyt bc 1- c y fusion complex supports the notion that structural super complexes between photosynthetic or respiratory components occur to ensure efficient cellular energy production.

Enhancing Stability and Oxidation Activity of Cytochrome c by Immobilization in the Nanochannels of Mesoporous Aluminosilicates

Hydrothermally stable and structrurally ordered mesoporous and microporous aluminosilicates with different pore sizes have been synthesized to immobilize cytochrome c (cyt c): MAS-9 (pore size 90 A), MCM-48-S (27 A), MCM-41-S (25 A), and Y zeolites (7.4 A). The amount of cyt c adsorption could be increased by the introduction of aluminum into the framework of pure silica materials. Among these mesoprous silicas (MPS), MAS-9 showed the highest loading capacity due to its large pore size. However, cyt c immobilized in MAS-9 could undergo facile unfolding during hydrothermal treatments. MCM-41-S and MCM-48-S have the pore sizes that match well the size of cyt c (25 x 25 x 37 A). Hence the adsorbed cyt c in these two medium pore size MPS have the highest hydrothermal stability and overall catalytic activity. On the other hand, the pore size of NaY zeolite is so small that cyt c is mostly adsorbed only on the outer surface and loses its enzymatic activity rapidly. The improved stability and high catalytic activity of cyt c immobilized in MPS are attributed to the electrostatic attraction between the pore surface and cyt c and the confinement provided by nanochannels. We further observed that cyt c immobilized in MPS exists in both high and low spin states, as inferred from the ESR and UV-vis studies. This is different from the native cyt c, which shows primarily the low spin state. The high spin state arises from the replacement of Met-80 ligands of heme Fe (III) by water or silanol group on silica surface, which could open up the heme groove for easy access of oxidants and substrates to iron center and facilitate the catalytic activity. In the catalytic study, MAS-9-cyt c showed the highest specific activity toward the oxidation of polycyclic aromatic hydrocarbons (PAHs), which arises from the fast mass transfer rate of reaction substrate due to its large pore size. For pinacyanol (a hydrophilic substrate), MCM-41-S-cyt c and MCM-48-S-cyt c showed higher specific activity than NaY-cyt c and MAS-9-cyt c. The result indicated that cyt c embedded in the channels of MCM-41-S and MCM-48-S was protected against unfolding and loss of activity. By increasing the concentration of the spin trapping agent, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in ESR experiments, we showed that cyt c catalyzes a homolytic cleavage of the O-O bond of hydroperoxide and generates a protein cation radical (g = 2.00). Possible mechanisms for MPS-cyt c catalytic oxidation of hydroperoxides and PAHs are proposed based on the spectroscopic characterizations of the systems.

Electrochemistry of Unfolded Cytochrome c in Neutral and Acidic Urea Solutions

The present investigation reports the first experimental measurements of the reorganization energy of unfolded metalloprotein in urea solution. Horse heart cytochrome c (cyt c) has been found to undergo reversible one-electron transfer reactions at pH 2 in the presence of 9 M urea. In contrast, the protein is electrochemically inactive at pH 2 under low-ionic strength conditions in the absence of urea. Urea is shown to induce ligation changes at the heme iron and lead to practically complete loss of the alpha-helical content of the protein. Despite being unfolded, the electron-transfer (ET) kinetics of cyt c on a 2-mercaptoethanol-modified Ag(111) electrode remain unusually fast and diffusion controlled. Acid titration of ferric cyt c in 9 M urea down to pH 2 is accompanied by protonation of one of the axial ligands, water binding to the heme iron (pK(a) = 5.2), and a sudden protein collapse (pH < 4). The formal redox potential of the urea-unfolded six-coordinate His18-Fe(III)-H(2)O/five-coordinate His18-Fe(II) couple at pH 2 is estimated to be -0.083 V vs NHE, about 130 mV more positive than seen for bis-His-ligated urea-denatured cyt c at pH 7. The unusually fast ET kinetics are assigned to low reorganization energy of acid/urea-unfolded cyt c at pH 2 (0.41 +/- 0.01 eV), which is actually lower than that of the native cyt c at pH 7 (0.6 +/- 0.02 eV), but closer to that of native bis-His-ligated cyt b(5) (0.44 +/- 0.02 eV). The roles of electronic coupling and heme-flattening on the rate of heterogeneous ET reactions are discussed.

Effect of Mono-CDNP Substitution of Lysine Residues on the Redox Reaction of Cytochrome c Electrostatically Adsorbed on a Mercaptoheptanoic Acid Modified Au(111) Surface

The effect of charge-inverting modification of single surface lysine residue on the electron transfer (ET) reaction of horse heart cytochrome c (cyt c) is examined for 12 different types of mono-4-chloro-2,5-dinitrobenzoic acid substituted cyt c (mCDNPc) adsorbed on a Au(111) electrode modified with a self-assembled monolayer (SAM) of 7-mercapto-heptanoic acid (MHA). A negative shift in the redox potential by 10-35 mV as compared to that of native cyt c and a monolayer coverage in the range of 13-17 pmol cm(-2) are observed for electroactive mCDNPc's. The magnitude of the decrease in the ET rate constant (k(et)) of mCDNPc's compared with that of native cyt c depends on the position of the CDNP substitution. For mCDNPc's in which the modified lysine residue is outside of the interaction domain of cyt c with the SAM, the ratio of the k(et) of mCDNPc to that of native cyt c is correlated to the change in the dipole moment vector of cyt c due to the CDNP modification. This correlation suggests that the dipole moment of cyt c determines its orientation of adsorption on the SAM of MHA and significantly affects the rate of the ET. The CDNP modification of lysine residues at the interaction domain significantly decreases the rate, demonstrating the importance of the local charge environment in determining the rate of ET.

Geminate carbon monoxide rebinding to a c-type haem

A chemically modified form of cytochrome c(cyt. c), termed carboxymethyl cytochrome c(cm cyt. c), possesses a vacant sixth coordination site to the haem iron that is available to bind external ligands. We present data on the rapid flash photolysis of CO from the ferrous haem iron of cm cyt. c and describe the kinetics and spectral transitions that accompany the recombination. This was achieved using 30-femtosecond laser pulses and a white light continuum to monitor spectral transitions. Whereas the photo-dissociation quantum yield is close to 1, the yield of CO escape from the protein (the apparent quantum yield, varphi) relative to myoglobin (varphi=1) is small due to rapid geminate recombination of CO. On ligand photo-dissociation the haem undergoes a spin-state transition from low-spin ferrous CO bound to penta-coordinate high-spin. Subsequently the system reverts to the CO bound form. The data were fitted with a minimum number of exponentials using global analysis. Recombination of CO with the haem iron of cm cyt. c is multiphasic (tau=16 ps, 120 ps and 1 ns), involving three spectrally distinct components. The fraction of haem (0.11) not recombining with CO within 4 ns is similar to the value of varphi(0.12) measured on the same preparation by the "pulse method" (M. Brunori, G. Giacometti, E. Antonini and J. Wyman, Proc. Natl. Acad. Sci. USA, 1973, 70, 3141-3144, ). This implies that no further geminate recombination occurs at t>4 ns. This unusually efficient CO-haem geminate recombination indicates the sterically hindered ("caged") nature of the distal haem pocket in cm cyt. c from which it is difficult for CO to escape. The large geminate phase may be contrasted with the behaviour of myoglobin in which geminate recombination is small. This is in general agreement with the well-documented extensive structural dynamics in myoglobin that allow ligand passage, and a higher structural rigidity in cyt. c imposed by the restraints of minimising reorganisation energy for electron transfer (M. Brunori, D. Bourgeois and D. Vallone, J. Struct. Biol., 2004, 147, 223-234, ). The high pH ferrous form of cm cyt. c is a low-spin species having a lysine bound to the central iron atom of the haem (M. Brunori, M. Wilson and E. Antonini, J. Biol. Chem., 1972, 247, 6076-6081; G. Silkstone, G. Stanway, P. Brzezinski and M. Wilson, Biophys. Chem., 2002, 98, 65-77, ). This high pH (pH approximately 8) form of deoxy cm cyt. c undergoes photo-dissociation of lysine (although the proximal histidine is possible) after photo-excitation. Recombination occurs with a time constant (tau) of approximately 7 ps. This is similar to that observed for the geminate rebinding of the Met80 residue in native ferrous cyt. c(tau approximately 6 ps) following its photo-dissociation (S. Cianetti, M. Negrerie, M. Vos, J.-L. Martin and S. Kruglik, J. Am. Chem. Soc., 2004, 126, 13 932-13 933; W. Wang, X. Ye, A. Demidov, F. Rosca, T. Sjodin, W. Cao, M. Sheeran and P. Champion, J. Phys. Chem., 2000, 104, 10 789-10 801, ).

The 40s ?-loop plays a critical role in the stability and the alkaline conformational transition of cytochrome c

The structural and redox properties of a non-covalent complex reconstituted upon mixing two non-contiguous fragments of horse cytochrome c, the residues 1-38 heme-containing N-fragment with the residues 57-104 C-fragment, have been investigated. With respect to native cyt c, the complex lacks a segment of 18 residues, corresponding, in the native protein, to an omega (Omega)-loop region. The fragment complex shows compact structure, native-like alpha-helix content but a less rigid atomic packing and reduced stability with respect to the native protein. Structural heterogeneity is observed at pH 7.0, involving formation of an axially misligated low-spin species and consequent partial displacement of Met80 from the sixth coordination position of the heme-iron. Spectroscopic data suggest that a lysine (located in the Met80-containing loop, namely Lys72, Lys73, or Lys79) replaces the methionine residue. The residues 1-38/57-104 fragment complex shows an unusual biphasic alkaline titration characterized by a low (p K(a1)=6.72) and a high p K(a)-associated state transition (p K(a2)=8.56); this behavior differs from that of native cyt c, which shows a monophasic alkaline transition (p K(a)=8.9). The data indicate that the 40s Omega-loop plays an important role in the stability of cyt c and in ensuring a correct alkaline conformational transition of the protein.

Ligand Binding Subsequent to NO Photolyses of Partially Unfolded Cytochrome c

AbstractHere we report folding studies of horse heart ferricytochrome c in the presence of NO (NO‐cyt c). The midpoint concentration of guanidine hydrochloride (GuHCl) for unfolding was measured to be 2.8 M in the presence of NO and is only moderately altered relative to native cyt c under similar conditions (i.e., 2.6 M GuHCl). Recombination of NO to cyt c in the presence of 2.8 M GuHCl subsequent to photolysis is biphasic with rate constants of (4.24 ± 0.15) × 103 s−1 and 197.7 ± 3.3 s−1 with the fast and slow phase representing ˜60% and 40% relative amplitudes, respectively. NO rebinding to unfolded cyt c (in the presence of 4 M GuHCl) exhibits two phases with rate constants similar to those observed in the presence of 2.8 M GuHCl with the relative amplitude of the fast phase increasing to roughly 90%. These data suggest that NO rebinds to two populations of cyt c with one population containing a five coordinate heme and the other having a six‐coordinate heme with bis‐His axial ligation. These results suggest that the mis‐ligated bis‐His conformation forms rapidly during the refolding of cyt. c3+, although a population exists that can refold down a native pathway.

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.

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.

Spin conversion of cytochrome b559 in photosystem II induced by exogenous high potential quinone

The spin-state of cytochrome b 559 (Cyt b 559 ) was studied in photosystem II (PSII) membrane fragments by low-temperature EPR spectroscopy. Treatment of the membranes with 2,3-dichloro-5,6-dicyano- p -benzoquinone (DDQ) converts the native low-spin (LS) form of Cyt b 559 to the high-spin (HS) form characterized with the g= 6.19 and g= 5.95 split signal. The HS Cyt b 559 was pH dependent with the amplitude increasing toward more acidic pH values (pH 5.5–8.5). The HS state was not photochemically active upon 77 and 200 K continuous illumination under our conditions and was characterized by a low reduction potential (⩽0 V). It was also demonstrated that DDQ treatment damages the oxygen evolving complex, leading to inhibition of oxygen evolution, decrease of the S 2 -state EPR multiline signal and release of Mn 2+ . In parallel, studies of model systems containing iron(III) protoporphyrin IX chloride (Fe III Por), which is a good model compound for the Cyt b 559 prosthetic group, were performed by using optical and EPR spectroscopy. The interaction of Fe III Por with imidazole (Im) in weakly polar solvent results in formation of bis -imidazole coordinated heme iron (Fe III Por Im 2 ) which mimic the bis -histidine axial ligation of Cyt b 559 . The reaction of DDQ with the LS Fe III Por Im 2 complex leads to its transformation into the HS state ( g ⊥ =5.95, g ∥ =2.00). It was shown that the spin conversion occurs due to the donor–acceptor interaction of coordinated imidazole with this high-potential quinone causing the displacement of imidazole from the axial position. The similar mechanism of DDQ-induced spin change is assumed to be valid for the native membrane Cyt b 559 in PSII centers.

Structural transformation of cytochrome c and apo cytochrome c induced by sulfonated polystyrene.

The structural transformation of cytochrome c (cyt c) and its heme-free precursor, apo cyt c, induced by negatively charged sulfonated polystyrene (SPS) with different charge density (degree of sulfonation) and chain length was studied to understand the factors that influence the folding and unfolding of the protein. SPS forms stable transparent nanoparticles in aqueous solution. The hydrophobic association of the backbone chain and phenyl groups is balanced by the electrostatic repulsion of the sulfonate groups on the particle surface. The binding of cyt c to negatively charged SPS particles causes an extensive disruption of the native compact structure of cyt c: the cleavage of Fe-Met80 ligand, about 40% loss of the helical structure, and the disruption of the asymmetry environment of Trp59. On the other hand, SPS particle-bound apo cyt c undergoes a conformational change from the random coil to alpha-helical structure. The folding of apo cyt c in SPS particles was influenced by pH and ionic strength of the solution, SPS concentration, and the degree of sulfonation and chain length of SPS. The folding can reach more than 90% of the alpha-helix content of native cyt c in solution. Poly(sodium 4-styrenesulfonate) (PSS), which is 100% sulfonated polystyrene and cannot form hydrophobic cores in the solution, induces only two-thirds of the alpha-helix content compared with SPS. It appears that the electrostatic interaction between PSS/SPS and apo cyt c induces an early partially folded state of apo cyt c. The hydrophobic interaction between nonpolar residues in apo cyt c and the hydrophobic cores in SPS particles extends the alpha-helical structure of apo cyt c.

Redox thermodynamics of cytochrome c in mixed water–organic solvent solutions

Bovine heart cytochrome c was studied through cyclic voltammetry in mixed water–organic solvent solutions under different conditions of temperature and the thermodynamic properties Δ S° rc and Δ H° rc calculated by the dependence of E° by temperature. The effect of the organic fraction of the solvent on the E° values of the native cyt c was found to be determined mainly by the decrease in dielectric constant of the medium. Specific interactions on the protein surface do not seem to play a remarkable role. The thermodynamic properties changes induced by the organic fraction have been interpreted tentatively in terms of solvation properties of cytochrome c and structural features of the protein environment.

Redox Properties of CytochromecAdsorbed on Self-Assembled Monolayers: A Probe for Protein Conformation and Orientation

The redox behavior of cytochrome c (cyt c) adsorbed to gold electrodes modified with self-assembled monolayers (SAMs) depends on the SAM. This paper examines SAMs generated from alkanethiols terminating in trimethylammonium (1), sulfonate (2), methyl (3), amine (4), and carboxylic acid (5) groups and from an aromatic thiol (6). The redox potentials of cyt c adsorbed on SAMs of 1 and 5 are relatively close to the formal potential of native cyt c measured in solution. The redox potentials of cyt c adsorbed on SAMs of 3, 4, and 6 are significantly shifted from the formal potential, and a reduction peak at about 0.5 V more negative than the formal potential (that is, a value corresponding to a more difficult reduction) was observed in all three cases. These observations suggest that cyt c changes its conformation significantly on adsorption on these surfaces. No redox peaks were observed for cyt c adsorbed on SAMs of 2, although surface plasmon resonance (SPR) studies indicate that the SAMs of 2 irreversibly adsorbed approximately a double layer of cyt c. Mixed SAMs were also studied. Most interestingly, cyt c adsorbed on mixed SAMs formed from the combinations of 1 and 2 exhibited significantly slower electron transfer (0.3−1.2 s-1) than cyt c adsorbed on a homogeneous SAM of 1 (45 s-1). These observations suggest changes in protein orientation due to the presence of the sulfonate groups at the interface. This study suggests that electrochemical measurement can be a useful probe for the conformation and orientation of protein adsorbed on surfaces.

Protein Oxidation of Cytochrome c by Reactive Halogen Species Enhances Its Peroxidase Activity

Reactive halogen species (RHS; X(2) and HOX, where X represents Cl, Br, or I) are metabolites mediated by neutrophil activation and its accompanying respiratory burst. We have investigated the interaction between RHS and mitochondrial cytochrome c (cyt c) by using electrospray mass spectrometry and electron spin resonance (ESR). When the purified cyt c was reacted with an excess amount of hypochlorous acid (HOCl) at pH 7.4, the peroxidase activity of cyt c was increased by 4.5-, 6.9-, and 8.6-fold at molar ratios (HOCl/cyt c) of 2, 4, and 8, respectively. In comparison with native cyt c, the mass spectra obtained from the HOCl-treated cyt c revealed that oxygen is covalently incorporated into the protein as indicated by molecular ions of m/z = 12,360 (cyt c), 12,376 (cyt c + O), and 12,392 (cyt c + 2O). Using tandem mass spectrometry, a peptide (obtained from the tryptic digests of HOCl-treated cyt c) corresponding to the amino acid sequence MIFAGIK, which contains the methionine that binds to the heme, was identified to be involved in the oxygen incorporation. The location of the oxygen incorporation was unequivocally determined to be the methionine residue, suggesting that the oxidation of heme ligand (Met-80) by HOCl results in the enhancement of peroxidase activity of cyt c. ESR spectroscopy of HOCl-oxidized cyt c, when reacted with H(2)O(2) in the presence of the nitroso spin trap 2-methyl-2-nitrosopropane (MNP), yielded more immobilized MNP/tyrosyl adduct than native cyt c. In the presence of H(2)O(2), the peroxidase activity of HOCl-oxidized cyt c exhibited an increasing ability to oxidize tyrosine to tyrosyl radical as measured directly by fast flow ESR. Titration of both native cyt c and HOCl-oxidized cyt c with various amounts of H(2)O(2) indicated that the latter has a decreased apparent K(m) for H(2)O(2), implicating that protein oxidation of cyt c increases its accessibility to H(2)O(2). HOCl-oxidized cyt c also displayed an impaired ability to support oxygen consumption by the purified mitochondrial cytochrome c oxidase, suggesting that protein oxidation of cyt c may break the electron transport chain and inhibit energy transduction in mitochondria.

A mammalian cytochrome fused to a chloroplast transit peptide is a functional haemoprotein and is imported into isolated chloroplasts

The small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a major chloroplast stromal protein that is cytosolically synthesized as a precursor with an N-terminal extension, known as the transit sequence or transit peptide (Tp). The Tp is essential for the post-translational uptake of the precursor by the chloroplast. The Tp is thought to influence the conformation of the precursor protein and to facilitate polypeptide translocation across the chloroplast envelope barrier via a Tp-selective translocon. To address these issues we have devised a novel strategy to generate substrate amounts of a chloroplast targeting sequence as a fusion with the chromogenic globular domain of cytochrome b(5) (Cyt). The chimaeric protein is an ideal probe for investigating the conformation of a preprotein and events surrounding protein import into isolated chloroplasts. The Cyt of liver endoplasmic reticulum was fused at its N-terminus with the Tp of the small subunit of Rubisco of Pisum sativum (pea). To enhance its production by clearance from the cytoplasm of Escherichia coli, the chimaera was engineered by further N-terminal linkage of a prokaryotic secretory signal. Expression of this tripartite fusion resulted in mg quantities of the signal sequence-processed Tp-Cyt protein, which was eventually targeted to the membranes. The chromogenic nature of the chimaera and its localization to the bacterial membrane facilitated the biochemical isolation of the precursor in a soluble and functional form. The purified preprotein displayed spectral and enzymic properties that were indistinguishable from the native parental Cyt, implying an absence of observable influence of the Tp on the conformation of the haemoprotein. The chimaeric precursor was imported into the stroma of the isolated chloroplasts in a dose-dependent manner. Import was also strongly dependent upon exogenously supplied ATP. The stromally imported chimaeric precursor protein was processed to a size characteristic of Cyt.

A mammalian cytochrome fused to a chloroplast transit peptide is a functional haemoprotein and is imported into isolated chloroplasts

The small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a major chloroplast stromal protein that is cytosolically synthesized as a precursor with an N-terminal extension, known as the transit sequence or transit peptide (Tp). The Tp is essential for the post-translational uptake of the precursor by the chloroplast. The Tp is thought to influence the conformation of the precursor protein and to facilitate polypeptide translocation across the chloroplast envelope barrier via a Tp-selective translocon. To address these issues we have devised a novel strategy to generate substrate amounts of a chloroplast targeting sequence as a fusion with the chromogenic globular domain of cytochrome b5 (Cyt). The chimaeric protein is an ideal probe for investigating the conformation of a preprotein and events surrounding protein import into isolated chloroplasts. The Cyt of liver endoplasmic reticulum was fused at its N-terminus with the Tp of the small subunit of Rubisco of Pisum sativum (pea). To enhance its production by clearance from the cytoplasm of Escherichia coli, the chimaera was engineered by further N-terminal linkage of a prokaryotic secretory signal. Expression of this tripartite fusion resulted in mg quantities of the signal sequence–processed Tp–Cyt protein, which was eventually targeted to the membranes. The chromogenic nature of the chimaera and its localization to the bacterial membrane facilitated the biochemical isolation of the precursor in a soluble and functional form. The purified preprotein displayed spectral and enzymic properties that were indistinguishable from the native parental Cyt, implying an absence of observable influence of the Tp on the conformation of the haemoprotein. The chimaeric precursor was imported into the stroma of the isolated chloroplasts in a dose-dependent manner. Import was also strongly dependent upon exogenously supplied ATP. The stromally imported chimaeric precursor protein was processed to a size characteristic of Cyt.

Unfolding and refolding of cytochrome c driven by the interaction with lipid micelles.

Binding of native cyt c to L-PG micelles leads to a partially unfolded conformation of cyt c. This micelle-bound state has no stable tertiary structure, but remains as alpha-helical as native cyt c in solution. In contrast, binding of the acid-unfolded cyt c to L-PG micelles induces folding of the polypeptide, resulting in a similar helical state to that originated from the binding of native cyt c to L-PG micelles. Far-ultraviolet (UV) circular dichroism (CD) spectra showed that this common micelle-associated helical state (HL) has a native-like alpha-helix content, but is highly expanded without a tightly packed hydrophobic core, as revealed by tryptophan fluorescence, near-UV, and Soret CD spectroscopy. The kinetics of the interaction of native and acid-unfolded cyt c was investigated by stopped-flow tryptophan fluorescence. Formation of H(L) from the native state requires the disruption of the tightly packed hydrophobic core in the native protein. This micelle-induced unfolding of cyt c occurs at a rate approximately 0.1 s(-1), which is remarkably faster in the lipid environment compared with the expected rate of unfolding in solution. Refolding of acid-unfolded cyt c with L-PG micelles involves an early highly helical collapsed state formed during the burst phase (<3 ms), and the observed main kinetic event reports on the opening of this early compact intermediate prior to insertion into the lipid micelle.

NMR studies of 4-methylimidazole binding to cytochrome c: effects of methyl substituent on the binding affinity, the orientation of the ligand plane, and the electronic structure of the heme †

The binding of 4-methylimidazole (mim) to cytochrome c (cyt c) has been studied by 1H NMR spectroscopy. The kinetic and thermodynamic parameters for the reaction were calculated. The assignment of a number of signals has led to the determination of the magnetic susceptibility tensor of mim–cyt c. It turned out that the orientation of the imidazole ring of mim was different from that of Him in Him–cyt c. This difference was due to the steric interaction between the 4-methyl and the surrounding peptides in the heme cavity. The pseudocontact and contact shifts of the four heme methyl groups in mim–cyt c were calculated. The hyperfine shift pattern and heme electron structure of mim–cyt c were compared with those of native cyt c and Him–cyt c.

Transient dimer in the refolding kinetics of cytochrome c characterized by small-angle X-ray scattering.

The equilibrium unfolding and the kinetic refolding of cytochrome c (Cyt c) in the presence of imidazole were studied with small-angle X-ray scattering (SAXS). The equilibrium unfolding experiments showed the radius of gyration, R(g), of native Cyt c to swell approximately 1 A with the addition of imidazole. The thermodynamic parameter m also reflects an expansion of the protein as its lower value demonstrates an increase in solvent-accessible surface area over that of native Cyt c in the absence of imidazole. Refolding was studied in the presence of imidazole as it prevents misligated intermediate states from forming during the refolding process, simplifying the kinetics, and making them easier to resolve. Time-resolved decreases in the forward scattering amplitude, I(0), demonstrated the transient formation of an aggregated intermediate. Final protein and denaturant concentrations were varied in the refolding kinetics, and the singular value decomposition (SVD) method was employed to characterize the associated state. This state was determined to be a dimer, with properties consistent with a molten globule.

Effects of solvent on the redox properties of cytochrome c: cyclic voltammetry and 1H NMR experiments in mixed water-dimethylsulfoxide solutions

Bovine heart cytochrome c (cyt c) was studied through cyclic voltammetry, 1H NMR and circular dichroism measurements in mixed water-dimethylsulfoxide (DMSO) solutions containing up to 50% DMSO by volume, under different conditions of temperature and pH. The effect of DMSO on the reduction potential of native cyt c was found to be determined mainly the he decrease in dielectric constant of the medium. No appreciable specific DMSO-protein interactions were detected. Instead, DMSO affects to some extent the conformation of alkaline cyt c and notably, stabilizes both redox states of this form to the detriment of the native form. In particular, DMSO lowers the p K a of the native to alkaline transition for oxidized cyt c and increases the electrochemical reversibility of the voltammetric wave of the alkaline form. DMSO-induced changes in the reduction entropy for native and alkaline cyt c were also determined and interpreted tentatively in terms of solvation properties of the heme and structural features of the protein environment.