A high redox potential form of cytochrome c550 in photosystem II from Thermosynechococcus elongatus

Cytochrome c(550) (cyt c(550)) is a component of photosystem II (PSII) from cyanobacteria, red algae, and some other eukaryotic algae. Its physiological role remains unclear. In the present work, measurements of the midpoint redox potential (E(m)) were performed using intact PSII core complexes preparations from a histidine-tagged PSII mutant strain of the thermophilic cyanobacterium Thermosynechococcus (T.) elongatus. When redox titrations were done in the absence of redox mediators, an E(m) value of +200 mV was obtained for cyt c(550). This value is ～300 mV more positive than that previously measured in the presence of mediators (E(m) = -80 mV). The shift from the high potential form (E(m) = +200 mV) to the low potential form (E(m) = -80 mV) of cyt c(550) is attributed to conformational changes, triggered by the reduction of a component of PSII that is sequestered and out of equilibrium with the medium, most likely the Mn(4)Ca cluster. This reduction can occur when reduced low potential redox mediators are present or under highly reducing conditions even in the absence of mediators. Based on these observations, it is suggested that the E(m) of +200 mV obtained without mediators could be the physiological redox potential of the cyt c(550) in PSII. This value opens the possibility of a redox function for cyt c(550) in PSII.     

Redox potential of cytochrome c550 in the cyanobacterium Thermosynechococcus elongates

Cytochrome c550 (cyt c550) from photosystem II (PSII) exists in the PSII-bound form but can be released from PSII by treatment with divalent cations or Tris, yielding the isolated form. We calculated heme redox potentials (Em) based on the crystal structures of cyt c550 by solving the Poisson-Boltzmann equation. In the isolated form, the calculated Em are -240 mV at pH 6.0 and -352 mV at pH 9.0. This pH-dependence is predominantly due to deprotonation of the heme-propionic group near Asn-49. In the PSII-bound form, the calculated E(m) was up-shifted by 160 mV versus the isolated form due to a conformational change of protein backbone, yielding Em=-84 mV.     

Factors that determine the unusually low reduction potential of cytochrome c 550 in cyanobacterial photosystem II

A new purification protocol for cytochrome c550 (cyt c550) from His-tagged SYnechocYstis PCC 6803 photosystem II (PSII) was developed which allows the protein to be isolated in high yield and purity. Electron paramagnetic resonance spectroscopy of cyt c550, both free in solution and in intact PSII preparations, yields identical spectra with g values at 1.50, 2.23, and 2.87, which are characteristic for a ferric low-spin bis-histidine coordinated heme. The resonance Raman spectrum of the isolated protein exhibits features characteristic of bis-histidine axial ligation of the iron and a slight ruffling of the heme macrocycle. Together, these results indicate that the heme structure is not very different from most c-type cytochromes, and thus the structure of the heme does not account for its unusually low reduction potential. A direct electrochemical measurement of the reduction potential was performed using square wave voltammetry on a pyrolytic graphite edge electrode, yielding E1.2=-108 mV (vs. NHE) with a peak separation of 5 mV. This value is 150 mV more positive than that previously measured by redox titrations. Because the behavior of the protein in the electrochemistry experiments is indicative of adsorption to the electrode surface, we surmise that binding of the protein to the electrode excludes solvent water from the heme-binding site. We conclude that the degree of solvent exposure makes a significant contribution to the heme reduction potential. Similarly, the binding of cyt c550 to PSII may also reduce the solvent exposure of the heme, and so the direct electrochemical value of the reduction potential may be relevant to the protein in its native state.     

Redox properties of the photosystem II cytochromes b559 and c550 in the cyanobacterium Thermosynechococcus elongatus

Redox properties of cytochrome b559 (Cyt b559) and cytochrome c550 (Cyt c550) have been studied by using highly stable photosystem II (PSII) core complex preparations from a mutant strain of the thermophilic cyanobacterium Thermosynechococcus elongatus with a histidine tag on the CP43 protein of PSII. Two different redox potential forms for Cyt b559 are found in these preparations, with a midpoint redox potential ( E'(m)) of +390 mV in about half of the centers and +275 mV in the other half. The high-potential form, whose E'(m)is pH independent, can be converted into the lower potential form by Tris washing, mild heating or alkaline pH incubation. The E'(m) of the low-potential form is significantly higher than that found in other photosynthetic organisms and is not affected by pH. The possibility that the heme of Cyt b559 in T. elongatus is in a more hydrophobic environment is discussed. Cyt c550 has a higher E'(m)when bound to the PSII core (-80 mV at pH 6.0) than after its extraction from the complex (-240 mV at pH 6.0). The E'(m) of Cyt c550 bound to PSII is pH independent, while in the purified state an increase of about 58 mV/pH unit is observed when the pH decreases below pH 9.0. Thus, Cyt c550 seems to have a single protonateable group which influences the redox properties of the heme. From these electrochemical measurements and from EPR controls it is proposed that important changes in the solvent accessibility to the heme and in the acid-base properties of that protonateable group could occur upon the release of Cyt c550 from PSII.     

Cytochrome c550 in the cyanobacterium Thermosynechococcus elongatus: study of redox mutants

Cytochrome c(550) is one of the extrinsic Photosystem II subunits in cyanobacteria and red algae. To study the possible role of the heme of the cytochrome c(550) we constructed two mutants of Thermosynechococcus elongatus in which the residue His-92, the sixth ligand of the heme, was replaced by a Met or a Cys in order to modify the redox properties of the heme. The H92M and H92C mutations changed the midpoint redox potential of the heme in the isolated cytochrome by +125 mV and -30 mV, respectively, compared with the wild type. The binding-induced increase of the redox potential observed in the wild type and the H92C mutant was absent in the H92M mutant. Both modified cytochromes were more easily detachable from the Photosystem II compared with the wild type. The Photosystem II activity in cells was not modified by the mutations suggesting that the redox potential of the cytochrome c(550) is not important for Photosystem II activity under normal growth conditions. A mutant lacking the cytochrome c(550) was also constructed. It showed a lowered affinity for Cl(-) and Ca(2+) as reported earlier for the cytochrome c(550)-less Synechocystis 6803 mutant, but it showed a shorter lived S(2)Q(B)(-) state, rather than a stabilized S(2) state and rapid deactivation of the enzyme in the dark, which were characteristic of the Synechocystis mutant. It is suggested that the latter effects may be caused by loss (or weaker binding) of the other extrinsic proteins rather than a direct effect of the absence of the cytochrome c(550).     

Extinction coefficients of cytochromes b559 and c550 of Thermosynechococcus elongatus and Cyt b559/PS II stoichiometry of higher plants

"Reduced minus oxidized" difference extinction coefficients Deltavarepsilon in the alpha-bands of Cyt b559 and Cyt c550 were determined by using functionally and structurally well-characterized PS II core complexes from the thermophilic cyanobacterium Thermosynechococcus elongatus. Values of 25.1+/-1.0 mM(-1) cm(-1) and 27.0+/-1.0 mM(-1) cm(-1) were obtained for Cyt b559 and Cyt c550, respectively. Anaerobic redox titrations covering the wide range from -250 up to +450 mV revealed that the heme groups of both Cyt b559 and Cyt c550 exhibit homogenous redox properties in the sample preparation used, with E(m) values at pH 6.5 of 244+/-11 mV and -94+/-21 mV, respectively. No HP form of Cyt b559 could be detected. Experiments performed on PS II membrane fragments of higher plants where the content of the high potential form of Cyt b559 was varied by special treatments (pH, heat) have shown that the alpha-band extinction of Cyt b559 does not depend on the redox form of the heme group. Based on the results of this study the Cyt b559/PSII stoichiometry is inferred to be 1:1 not only in thermophilic cyanobacteria as known from the crystal structure but also in PSII of plants. Possible interrelationships between the structure of the Q(B) site and the microenvironment of the heme group of Cyt b559 are discussed.     

In situ effects of mutations of the extrinsic cytochrome c550 of photosystem II in Synechocystis sp. PCC6803

The H(2)O oxidizing domain of the cyanobacterial photosystem II (PSII) complex contains a low potential, c-type cytochrome termed c(550) that is essential for the in vivo stability of the PSII complex. A mutant lacking cytochrome c(550) (DeltapsbV) in Synechocystis sp. PCC6803 has been further analyzed together with a construct in which the distal axial heme iron ligand, histidine 92, has been substituted with a methionine (C550-H92M). Heme staining of SDS-PAGE showed that the C550-H92M mutation did not disturb the accumulation and heme-binding properties of the cytochrome. In DeltapsbV cells, the number of charge separating PSII centers was estimated to be 56% of the wild type, but of the existing centers, 33% lacked photooxidizable Mn ions. C550-H92M did not discernibly affect the intrinsic PSII electron-transfer kinetics compared to the wild type nor did it exhibit a significant fraction of centers lacking photooxidizable Mn; however, the number of charge separating PSII centers in mutant cells was 69% of the wild type. C550-H92M lost photoautotrophic growth ability in the absence of Ca(2+), but its growth was not affected by depletion of Cl(-), which differs from DeltapsbV. Taken together, the results suggest that in the absence of cytochrome c(550) electron transfer on the donor side is retarded perhaps at the level of Y(z) to P680(+) transfer, the heme ligand. His92 is not absolutely required for assembly of functional PSII centers; however, replacement by methionine prevents normal accumulation of PSII centers in the thylakoid membranes and alters the Ca(2+) requirement of PSII. The results are discussed in terms of current understanding of the Ca(2+) site of PSII.     

Water-splitting manganese complex controls light-induced redox changes of cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>559</Subscript> in Photosystem II

The effect of water-splitting Mn complex on light-induced redox changes of cytochrome b ₅₅₉ (cyt b ₅₅₉) was studied in spinach photosystem II (PSII) membranes. Photoreduction of the heme iron in the intact PSII membranes was completely suppressed by DCMU, whereas photoreduction and photooxidation of the heme iron in the Mn-depleted PSII membranes were unaffected by DCMU. Interesingly, photoreduction and photooxidation of the heme iron in the Mn-depleted PSII membranes were completely diminished by exogenous superoxide dismutase (SOD), whereas no effect of SOD on photoreduction of the heme iron was observed in the intact PSII membranes. The current work shows that the light-induced redox changes of cyt b ₅₅₉ proceed via a different mechanism in the both types of PSII membranes. In the intact PSII membranes, photoreduction of the heme iron is mediated by plastoquinol. However, in the Mn-depleted PSII membranes, photoreduction and photooxidation of the heme iron are mediated by superoxide anion radical formed in PSII.     

Characterization of the multiple forms of cytochrome b559 in photosystem II

Cytochrome b559 is an essential component of the photosystem II (PSII) protein complex. Its function, which has long been an unsolved puzzle, is likely to be related to the unique ability of PSII to oxidize water. We have used EPR spectroscopy and spectrophotometric redox titrations to probe the structure of cytochrome b559 in PSII samples that have been treated to remove specific components of the complex. The results of these experiments indicate that the low-temperature photooxidation of cytochrome b559 does not require the presence of the 17-, 23-, or 33-kDa extrinsic polypeptides or the Mn complex (the active site in water oxidation). We observe a shift in the g value of the EPR signal of cytochrome b559 upon warming a low-temperature photooxidized sample, which presumably reflects a change in conformation to accommodate the oxidized state. At least three redox forms of cytochrome b559 are observed. Untreated PSII membranes contain one high-potential (375 mV) and one intermediate-potential (230 mV) cytochrome b559 per PSII. Thylakoid membranes also appear to contain one high-potential and one intermediate-potential cytochrome b559 per PSII, although this measurement is more difficult due to interference from other cytochromes. Removal of the 17- and 23-kDa extrinsic polypeptides from PSII membranes shifts the composition to one intermediate-potential (170 mV) and one low-potential (5 mV) cytochrome b559. This large decrease in potential is accompanied by a very small g-value change (0.04 at gz), indicating that it is the environment and not the ligand field of the heme which changes significantly upon the removal of the 17- and 23-kDa polypeptides.     

Strategic roles of axial histidines in structure formation and redox regulation of tetraheme cytochrome c3

Tetraheme cytochrome c 3 (cyt c 3) exhibits extremely low reduction potentials and unique properties. Since axial ligands should be the most important factors for this protein, every axial histidine of Desulfovibrio vulgaris Miyazaki F cyt c 3 was replaced with methionine, one by one. On mutation at the fifth ligand, the relevant heme could not be linked to the polypeptide, revealing the essential role of the fifth histidine in heme linking. The fifth histidine is the key residue in the structure formation and redox regulation of a c-type cytochrome. A crystal structure has been obtained for only H25M cyt c 3. The overall structure was not affected by the mutation except for the sixth methionine coordination at heme 3. NMR spectra revealed that each mutated methionine is coordinated to the sixth site of the relevant heme in the reduced state, while ligand conversion takes place at hemes 1 and 4 during oxidation at pH 7. The replacement of the sixth ligand with methionine caused an increase in the reduction potential of the mutated heme of 222-244 mV. The midpoint potential of a triheme H52M cyt c 3 is higher than that of the wild type by approximately 50 mV, suggesting a contribution of the tetraheme architecture to the lowering of the reduction potentials. The hydrogen bonding of Thr24 with an axial ligand induces a decrease in reduction potential of approximately 50 mV. In conclusion, the bis-histidine coordination is strategically essential for the structure formation and the extremely low reduction potential of cyt c 3.     

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.     

Inorganic cofactor stabilization and retention: the unique functions of the two PsbO subunits of eukaryotic photosystem II

Eukaryotic PsbO, the photosystem II (PSII) manganese-stabilizing protein, has two N-terminal sequences that are required for binding of two copies of the protein to PSII [Popelkova, H., et al. (2002) Biochemistry 41, 10038-10045; Popelkova, H., et al. (2003) Biochemistry 42, 6193-6200]. In the work reported here, a set of selected N-terminal truncation mutants of PsbO that affect subunit binding to PSII were used to determine the effects of PsbO stoichiometry on the Mn, Ca(2+), and Cl(-) cofactors and to characterize the roles of each of the PsbO subunits in PSII function. Results of the experiments with the PsbO-depleted PSII membranes reconstituted with the PsbO deletion mutants showed that the presence of PsbO does not affect Ca(2+) retention by PSII in steady-state assays of activity, nor is it required for Ca(2+) to protect the Mn cluster against reductive inhibition in darkness. In contrast to the results with Ca(2+), PsbO increases the affinity of Cl(-) for the active site of the O(2)-evolving complex (OEC) as expected. These results together with other data on activity retention suggest that PsbO can stabilize the Mn cluster by facilitating retention of Cl(-) in the OEC. The data presented here indicate that each of two copies of PsbO has a distinctive function in PSII. Binding of the first PsbO subunit fully stabilizes the Mn cluster and enhances Cl(-) retention, while binding of the second subunit optimizes Cl(-) retention, which in turn maximizes O(2) evolution activity. Nonspecific binding of some PsbO truncation mutants to PSII has no functional significance.     

The Psb27 protein facilitates manganese cluster assembly in photosystem II

Photosystem II (PSII) is a large membrane protein complex that uses light energy to convert water to molecular oxygen. This enzyme undergoes an intricate assembly process to ensure accurate and efficient positioning of its many components. It has been proposed that the Psb27 protein, a lumenal extrinsic subunit, serves as a PSII assembly factor. Using a psb27 genetic deletion strain (Deltapsb27) of the cyanobacterium Synechocystis sp. PCC 6803, we have defined the role of the Psb27 protein in PSII biogenesis. While the Psb27 protein was not essential for photosynthetic activity, various PSII assembly assays revealed that the Deltapsb27 mutant was defective in integration of the Mn(4)Ca(1)Cl(x) cluster, the catalytic core of the oxygen-evolving machinery within the PSII complex. The other lumenal extrinsic proteins (PsbO, PsbU, PsbV, and PsbQ) are key components of the fully assembled PSII complex and are important for the water oxidation reaction, but we propose that the Psb27 protein has a distinct function separate from these subunits. We show that the Psb27 protein facilitates Mn(4)Ca(1)Cl(x) cluster assembly in PSII at least in part by preventing the premature association of the other extrinsic proteins. Thus, we propose an exchange of lumenal subunits and cofactors during PSII assembly, in that the Psb27 protein is replaced by the other extrinsic proteins upon assembly of the Mn(4)Ca(1)Cl(x) cluster. Furthermore, we show that the Psb27 protein provides a selective advantage for cyanobacterial cells under conditions such as nutrient deprivation where Mn(4)Ca(1)Cl(x) cluster assembly efficiency is critical for survival.     

Near-infrared-induced transitions in the manganese cluster of photosystem II: action spectra for the S2 and S3 redox states

The Mn4Ca complex that is involved in water oxidation in PSII is affected by near-infrared (NIR) light in certain redox states and these phenomena can be monitored by electron paramagnetic resonance (EPR) at low temperature. Here we report the action spectra of the NIR effects in the S2 and S3 states in PSII from plants and the thermophilic cyanobacterium Thermosynechococcus elongatus. The action spectra obtained are very similar in both S states, indicating the presence of the same photoactive form of the Mn4Ca complex in both states. Since the chemical nature of the photoactive species is not known, an unequivocal interpretation of this result cannot be made; however, it appears to be more easily reconciled with the view that the redox state of the Mn4Ca cluster does not change from the S2 to the S3 transition, at least in those centers sensitive to NIR light. The temperature dependence of the NIR effect and the action spectra for S2 indicate the presence of structural heterogeneity in the Mn4Ca cluster.     

Influence of the PsbA1/PsbA3, Ca(2+)/Sr(2+) and Cl(-)/Br(-) exchanges on the redox potential of the primary quinone Q(A) in Photosystem II from Thermosynechococcus elongatus as revealed by spectroelectrochemistry

Ca(2+) and Cl(-) ions are essential elements for the oxygen evolution activity of photosystem II (PSII). It has been demonstrated that these ions can be exchanged with Sr(2+) and Br(-), respectively, and that these ion exchanges modify the kinetics of some electron transfer reactions at the Mn?Ca cluster level (Ishida et al., J. Biol. Chem. 283 (2008) 13330-13340). It has been proposed from thermoluminescence experiments that the kinetic effects arise, at least in part, from a decrease in the free energy level of the Mn(4)Ca cluster in the S? state though some changes on the acceptor side were also observed. Therefore, in the present work, by using thin-layer cell spectroelectrochemistry, the effects of the Ca(2+)/Sr(2+) and Cl(-)/Br(-) exchanges on the redox potential of the primary quinone electron acceptor Q(A), E(m)(Q(A)/Q(A)(-)), were investigated. Since the previous studies on the Ca(2+)/Sr(2+) and Cl(-)/Br(-) exchanges were performed in PsbA3-containing PSII purified from the thermophilic cyanobacterium Thermosynechococcus elongatus, we first investigated the influences of the PsbA1/PsbA3 exchange on E(m)(Q(A)/Q(A)(-)). Here we show that i) the E(m)(Q(A)/Q(A)(-)) was up-shifted by ca. +38mV in PsbA3-PSII when compared to PsbA1-PSII and ii) the Ca(2+)/Sr(2+) exchange up-shifted the E(m)(Q(A)/Q(A)(-)) by ca. +27mV, whereas the Cl(-)/Br(-) exchange hardly influenced E(m)(Q(A)/Q(A)(-)). On the basis of the results of E(m)(Q(A)/Q(A)(-)) together with previous thermoluminescence measurements, the ion-exchange effects on the energetics in PSII are discussed.     

Detection of an electron paramagnetic resonance signal in the S0 state of the manganese complex of photosystem II from Synechococcus elongatus.

The Mn(4)-cluster of photosystem II (PSII) from Synechococcus elongatus was studied by electron paramagnetic resonance (EPR) spectroscopy after a series of saturating laser flashes given in the presence of either methanol or ethanol. Results were compared to those obtained in similar experiments done on PSII isolated from plants. The flash-dependent changes in amplitude of the EPR multiline signals were virtually identical in all samples. In agreement with earlier work [Messinger, J., Nugent, J. H. A., and Evans, M. C. W. (1997) Biochemistry 36, 11055-11060; Ahrling, K. A., Peterson, S., and Styring, S. (1997) Biochemistry 36, 13148-13152], detection of an EPR multiline signal from the S(0) state in PSII from plants was only possible with methanol present. In PSII from S. elongatus, it is shown that the S(0) state exhibits an EPR multiline signal in the absence of methanol (however, ethanol was present as a solvent for the artificial electron acceptor). The hyperfine lines are better resolved when methanol is present. The S(0) multiline signals detected in plant PSII and in S. elongatus were similar but not identical. Unlike the situation seen in plant PSII, the S(2) state in S. elongatus is not affected by the addition of methanol in that (i) the S(2) multiline EPR signal is not modified by methanol and (ii) the spin state of the S(2) state is affected by infrared light when methanol is present. It is also shown that the magnetic relaxation properties of an oxidized low-spin heme, attributed to cytochrome c(550), vary with the S states. This heme then is in the magnetic environment of the Mn(4) cluster.     

Structural and functional modulation of the manganese cluster in Ca(2+)-depleted photosystem II induced by binding of the 24-kilodalton extrinsic protein.

Depletion of functional Ca2+ from photosystem (PS) II membranes impairs O2 evolution. Redox properties of the Mn cluster as probed by thermoluminescence were modified differently in Ca(2+)-depleted PSII depending on the procedure for Ca2+ extraction. Ca2+ depletion by low-pH treatment gave rise to an abnormally modified S2 state exhibiting a thermoluminescence band with elevated peak temperature accompanied by a marked upshift in threshold temperature for its formation, whereas Ca2+ depletion by NaCl washing in the light followed by the addition of EDTA could generate a similarly modified S2 state only when the Ca(2+)-depleted PSII was reconstituted with the 24-kDa extrinsic proteins. These results indicated that manifestation of the abnormal properties of the Ca(2+)-depleted S2 state is significantly contributed by the association of the 24-kDa extrinsic protein to PSII. It was inferred that the 24-kDa extrinsic protein regulates the structure and function of the Mn cluster in the absence of functional Ca2+ through a conformational modulation of the intrinsic protein(s) that bind(s) both Mn and Ca. Features of the extrinsic protein-dependent modulation of the Mn cluster were discussed in relation to the function of Ca2+ in O2 evolution.     

Extinction coefficients of cytochromes b559 and c550 of Thermosynechococcus elongatus and Cyt b559/PS II stoichiometry of higher plants

“Reduced minus oxidized” difference extinction coefficients Δ? in the α-bands of Cyt b559 and Cyt c550 were determined by using functionally and structurally well-characterized PS II core complexes from the thermophilic cyanobacterium Thermosynechococcus elongatus. Values of 25.1 ± 1.0 mM⁻¹ cm⁻¹ and 27.0 ± 1.0 mM⁻¹ cm⁻¹ were obtained for Cyt b559 and Cyt c550, respectively. Anaerobic redox titrations covering the wide range from −250 up to +450 mV revealed that the heme groups of both Cyt b559 and Cyt c550 exhibit homogenous redox properties in the sample preparation used, with E_m values at pH 6.5 of 244 ± 11 mV and −94 ± 21 mV, respectively. No HP form of Cyt b559 could be detected. Experiments performed on PS II membrane fragments of higher plants where the content of the high potential form of Cyt b559 was varied by special treatments (pH, heat) have shown that the α-band extinction of Cyt b559 does not depend on the redox form of the heme group. Based on the results of this study the Cyt b559/PSII stoichiometry is inferred to be 1:1 not only in thermophilic cyanobacteria as known from the crystal structure but also in PSII of plants. Possible interrelationships between the structure of the Q_B site and the microenvironment of the heme group of Cyt b559 are discussed.     

How does reorganization energy change upon protein unfolding? Monitoring the structural perturbations in the heme cavity of cytochrome c

In several classes of proteins the redox center provides an additional intrinsic biophysical probe that could be used to study the protein structure and function. In present report reorganization energy (lambda, as a parameter describing electron transfer properties) was used to study the protein structural changes around the heme prosthetic group in cytochrome c (cyt c). We attempted to monitor the value of this parameter upon the unfolding process of cyt c by urea, during which it was increased sigmoidally from about 0.52 to 0.82 eV for native and unfold protein, respectively. Results indicate that by structural changes in the heme site, lambda provides a complementary tool for following the unfolding process. Assuming a reversible two-state model for cyt c unfolding, Delta G(H2O), Cm and m values were determined to be 8.32+/-0.7 kcal mol(-1), 1.53+/-0.19 kcalmol(-1)M(-1) and 5.03 M, respectively.     

Characterization of a new membrane-bound cytochrome c of Rhodopseudomonas capsulata

A cytochrome c (cyt. c) was solubilized with Triton-X-100 and co-purified with cytochrome c oxidase from membranes of chemotrophically grown cells of Rhodopseudomonas capsulata. Cyt. c and cytochrome oxidase were separated on Sephadex G-50 columns. Antibodies against cytochrome c2 from the same bacterium did not cross react with the membrane-bound cyt. c. The IEP of the membrane-bound cyt. c was found to be pH 8.2, the midpoint potential was 234 +/- 11 mV at pH 7.0. This cyt. c binds CO. The native cyt. c is a dimer with an apparent Mr of 25000 containing 2 mol heme per mol dimer, which is believed to function as an electron donor for the high-potential cytochrome c oxidase.