| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
* Institute for Protein Research, Osaka University, Suita, Japan; Faculty of Engineering, Yokohama National University, Yokohama, Japan; Yokohama Research & Development Center, Mitsubishi Heavy Industries, Yokohama, Japan; Department of Biochemistry, University of Arizona, Tucson, Arizona; ¶ Division of Chemistry, Graduate School of Science, Kyoto University, Kyoto, Japan; || RIKEN Harima Institute/SPring-8, Hyogo, Japan; and ** Institute for Molecular Science, Okazaki National Institutes, Okazaki, Japan
Correspondence: Address reprint requests to Hideo Akutsu, Osaka University, 3-2 Yamadaoka, Suita 565-0871, Japan. Tel.: 81-6-6879-8597; Fax: 81-6-6879-8599; E-mail: akutsu{at}protein.osaka-u.ac.jp.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
|---|
) from the sulfate-reducing bacteria have been isolated and characterized. Within the superfamily (Bruschi, 1994), examples include tetraheme cytochromes c3 (cyt c3; mol. wt. 
14,000; Coutinho and Xavier, 1994), octaheme cyt c3 (mol. wt. 26,000; Bruschi, 1994), and hexadecaheme cytochrome, Hmc (Pollock et al., 1991). The three dimensional structures of several tetraheme cyt c3 have been determined (Harada et al., 2002 and references therein), as well as the structures of octaheme cyt c3 (Czjzek et al., 1996) and Hmc (Matias et al., 2002; Czjzek et al., 2002). An important characteristic of cyt c3 is the extremely low oxidation-reduction (redox) potential of the hemes, ranging from -165 to -400 mV as compared to 260 mV for the mitochondrial Class I c-type cytochromes (Mathews, 1985). The low redox potential hemes, a consequence of bis-histidine ligation and the environment provided by the folded protein, facilitates the metabolic need for the Desulfovibrio to reduce the relatively weak oxidant, sulfate. In the course of studies designed to better understand the folding, redox properties and function of the cyt c3, site-directed mutagenesis using an overexpression system from Desulfovibrio desulfricans has been effectively used (Dolla et al., 1994; Salgueiro et al., 2001). Nevertheless, much remains to be learned about the role of specific amino-acid side chains, in part because of limited amounts of mutated cytochrome. Recently, we have developed a novel overexpression system of multiheme cyt c3 using the facultative anaerobe, Shewanella oneidensis (Ozawa et al., 2001). This system permits the production of relatively large amounts of cyt c3 site-directed mutants and thus facilitates structural studies.
One of the important characteristics of cyt c3 is the presence of aromatic rings in the vicinity of axial ligands of four hemes. The significance of these aromatic rings has been indicated in the literature from the structural point of view (Higuchi et al., 1984; Czjzek et al., 1994), although their individual roles are still not clear. Among the highly conserved aromatic residues, tyrosine 43 (Y43) is an interesting candidate for structural studies. It is present in Desulfovibrio vulgaris Miyazaki F (DvMF), D. vulgaris Hildenborough (DvH), D. gigas (Dg), and D. desulfuricans ATCC 27774 (DdA) (Nørager et al., 1999), which commonly have the heme binding motifs CXXCH, CXXXXCH, CXXCH, and CXXXXCH from the amino to carboxyl termini (X is any amino acid). The aromatic ring of this tyrosine is parallel to the imidazole of histidine 34, the fifth axial ligand of heme 1 (the heme numbering according to the sequence) and in close proximity (within 4 Å). Note that the DvMF cyt c3 has one more alanine at the N-terminus (Ozawa et al., 2001) compared to the reported sequence (Kitamura et al., 1993). We will generally use the originally reported sequence numbering to retain consistency with the literature. When the new numbering is required, it will be shown in italics.
We have focused our attention on Y43, and have investigated the structural and redox properties of two mutations, Y43F and Y43L. Moreover, the kinetics of reduction of Y43L cyt c3 by 5-deazariboflavin semiquinone (5-dRfH·, Em = -650 mV) were also investigated using laser flash photolysis to examine the effect of amino-acid replacement on electron transfer kinetics. The physicochemical and kinetic results obtained are discussed in the context of the three-dimensional structure of Y43L cyt c3 at 0.91 Å resolution.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
|---|
).
Site-directed mutagenesis and purification of mutant proteins
Plasmid pUCMC3, containing the gene for DvMF cyt c3 (Ozawa et al., 2001), was digested with PstI and EcoRI, and the DvMF cyt c3 gene was ligated at the same sites of the pKF19k vector (Gene bank accession No. D63847) to generate pKFC3k. Plasmid pKFC3k containing dual-amber mutations on the gene encoding a kanamycin resistance protein was used as a template for site-directed mutagenesis. The codon for Y43 in the cyt c3 gene in pKFC3k was replaced with that for Leu or Phe, using oligonucleotide-dual-amber-LA PCR-based mutagenesis, essentially as described by Hashimoto-Gotoh et al. (1995). Mutagenic oligonucleotides Y43L (5'-CGGCAAGGAAGATCTCCAGAAGTGCGCC-3') and Y43F (5'-CGGCAAGGAAGATTTCCAGAAGTGCGCC-3') were used to change the TAC codon at nucleotides 459–461 (Kitamura et al., 1993) into CTC and TTC, respectively.
The mutated genes were sequenced with a LI-COR Model 4200 DNA sequencer to confirm the presence of the desired mutations and the absence of any unwanted mutations. The mutated plasmids, pY43L and pY43F, were directly electrotransferred to Shewanella oneidensis TSP-C (Ozawa et al., 2001), and the recombinants were aerobically grown at 30°C in two liters of 2 x YT (with 10 mg rifampicin and 200 mg kanamycin /L) media in 3-liter Erlenmeyer flasks. The mutant cyt c3 were purified according to the reported method (Ozawa et al., 2000). Protein mass numbers were determined by MALDI-TOF mass spectrometry using a Voyager TMDE (PerSeptive Biosystems, Framingham, MA) to confirm the mutation.
NMR and electrochemical characterization of the mutant DvMF cyt c3
For NMR measurements, samples (12 mM) were prepared as previously described (Park et al., 1996). Most 1H-NMR spectra were recorded at 303 K in 30 mM sodium phosphate buffer (2H2O), at p2H 7.0, at 400, 500, and 600 MHz with Bruker (Karlsruhe, Germany), DRX-400, DRX-500, and DRX-600 spectrometers respectively. NOESY spectra were measured with a mixing time of 15 ms, a data size of 2048 x 512, and a spectral width of 24 kHz. Chemical shifts are presented in parts per million (ppm) relative to the internal standard of 2,2-dimethyl-2-silapentane-5-sulfonate (DSS). Differential pulse polarograms were obtained with a PerkinElmer (Oak Ridge, TN) 394 digital electrochemical trace analysis system using a dropping mercury electrode. The modulation amplitude, sweep rate, and drop time were 20 mV, 4 mVs-1, and 2 s, respectively. The polarograms were fitted using the analytical equation for the four consecutive one-electron reversible electrode reactions (Niki et al., 1984). Redox potentials are referred to the standard hydrogen electrode at 30°C in this work.
Laser flash photolysis experiments
Laser flash photolysis was carried out at room temperature using an N2-dye laser (Laser photonics), which excites 5-deazariboflavin (5-dRf) to produce its semiquinone species, 5-dRfH· (Em = -650 mV) in the presence of excess EDTA (Tollin et al., 1986). Then 5-dRfH· reduces cyt c3. The concentrations of 5-dRf and EDTA were 60–120 µM and 10 mM, respectively. All kinetic experiments were performed under pseudo first-order conditions with a heme concentration of 2–5 µM. The buffer solution (20 mM sodium phosphate, pH 7) was placed in a rubber septum-sealed cuvette and bubbled with oxygen-depleted and water-saturated argon for 1 h to remove the residual oxygen before addition of cytochrome. The reduction of cyt c3 by 5-dRfH· was monitored as the intensity of the 
-peak at 552 nm. Before laser flash photolysis, the cyt c3 was partially reduced by illuminating the sample in the presence of 5-dRfH· and EDTA, with a 35-W tungsten lamp at a distance of 12 cm for illumination with 1–30 s intervals. The concentration of oxidized heme was determined spectrophotometrically before each laser flash, and was checked immediately after the flash photolysis to ensure that the concentration of the oxidized heme had not changed significantly.
Crystallization and three-dimensional structure determination of wild-type and Y43L cyt c3
Crystals of the wild-type and Y43L cyt c3 from DvMF were grown at 10°C by the vapor diffusion method. Forty to 100 µl of the protein solution, 15 mg/ml (12.5 mM Tris-HCl, pH 7.4) containing 50% (v/v) ethanol, was equilibrated against 10 ml of a buffer solution (10 mM Tris-HCl, pH 7.4) containing 60%(v/v) ethanol. Diffraction experiments were carried out at 100 K using synchrotron x-ray beams (wavelengths: 0.700 Å, Y43L; and 0.710 Å, wild-type) at the BL-44B2 beam line of SPring-8. The crystals of the wild-type and Y43L cyt c3 belong to orthorhombic space group P212121. The changes in the cell parameters between two structures were within 1%. Since the crystals diffracted to the ultrahigh resolution range, diffraction data in low (
= 1.80 Å) and high ( = 0.90 Å) resolution ranges were separately collected under different conditions, using a MAR-CCD detector system. They were processed and merged with programs MOSFLM (Leslie, 1990) and SCALA (Collaborative Computational Project, 1994), respectively. The structure refinement was started with a program package of X-PLOR (Brünger, 1992) using the atomic coordinates of the wild-type cyt c3 at 1.8 Å resolution (Higuchi et al., 1984) without water molecules, and followed with SHELXL (Sheldrick and Schneider, 1997). All atoms including water oxygen atoms were refined with anisotropic B factors. At the final stage of the refinement, hydrogen atoms were incorporated at the calculated positions.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
|---|
|
|
|
, Fan et al., 1990). The individual spectra correspond to those of the five macroscopic oxidation states: the fully-oxidized (S0), the one-electron-reduced (S1), the two-electron-reduced (S2), the three-electron-reduced (S3), and the fully-reduced (S4) states. This observation can be explained by the combination of two distinct exchange rates: first, by the intermolecular electron exchange that is sufficiently slow to give rise to distinct signals for each macroscopic oxidation state; and second, by the intramolecular electron exchange between hemes within cyt c3, which is sufficiently fast to average out the chemical shifts of each heme for the oxidized and reduced states, giving rise to single set of signals for each heme in a certain macroscopic oxidation state. However, line-widths of the spectra of Y43L cyt c3 in the intermediate oxidation states are broader than those of the wild type and Y43F (30–150 Hz). Since the line-width is the function of ratio of the exchange rate to the chemical shift difference in two oxidation states, either the intramolecular or the intermolecular electron exchange is affected by the Y43L mutation. In the former case, the line broadening will become more significant on lowering the temperature (that is, slowing the exchange rate), whereas the situation is the opposite in the latter case. As can be seen in Fig. 3, the line-widths of the S1 signals become sharper with an increase in temperature (from 330 Hz at 283 K to 133 Hz at 303 K for signal B), suggesting that the origin of the broadening is the slower intramolecular electron transfer. This conclusion is confirmed by the observation that the spectrum for the mixture of the S0 and S1 states shows that the line-widths of the signals for S1 are much broader than those for S0 (Fig. 3), because the intermolecular exchange broadening should also appear in the signals in S0. Line-broadenings of the heme methyl signals were reported in the intermediate oxidation states for DvH cyt c3 (Turner et al., 1996). They were attributed to the slow proton exchange because of different pKa in different oxidation states. This is not the case for Y43L cyt c3, because the origin of the line-broadening is the fast exchange as mentioned above.
|
). The assignment of the heme methyl signals in the five macroscopic oxidation states were established on the basis of the assignment in the fully reduced state. The results for Y43L and Y43F cytochromes c3 are summarized in Table 1. The reduction fraction (R) of each heme (the contribution to total reduction of each heme) can be obtained from the chemical shifts in Table 1 according to the equation in Table 2 (Fan et al., 1990). The average reduction fractions were calculated under the conditions of 
using signals B for heme 1, C for heme 2, E for heme 3, and H for heme 4, following the selection of signals by Turner et al. (1996), and given in Table 2. Here, i and j stand for the heme and reduction step numbers, respectively. The data in Table 2 demonstrates that the order of reduction (major fraction at each step) is hemes 4, 1, 2, and 3, for both Y43L and Y43F cyt c3, as has been found for the wild type (Park et al., 1996).
|
). The results are presented in Table 3 together with the results for the wild type. In the Y43L mutation, the changes in e1 are relatively large (44 and 34 mV) in comparison with those in other hemes, showing that the effect of mutation is local. It led to inversion of the order of 
and 
With the Y43F mutation, however, the changes are small for every heme.
|
). An insight into the contributions of the individual hemes to the rate constant was obtained by performing photo titration experiments involving laser flash photolysis on cyt c3 samples that were partially reduced by white light illumination before flash photolysis. The pseudo first-order rate constant (kobs) for reduction by 5-dRfH· was plotted against the fraction of the heme reduced before the laser flash, as shown in Fig. 4. The observed curve is quite different for the wild type as compared to the Y43L mutant. To obtain information on the individual rate constant for each macroscopic oxidation form (the molecules in the Si state), the kinetic data can be analyzed assuming that the reduction of each macroscopic oxidation form makes a contribution to the observed kinetics that depends on its rate constant and the relative amount of the heme oxidized (Akutsu et al., 1992). The concentrations of the fully oxidized, one-electron-reduced, two-electron-reduced, and three-electron-reduced forms can be estimated using the macroscopic redox potentials and the total concentrations of the oxidized hemes obtained from the absorption spectrum.
|
),
 | (1a) |
 | (1b) |
 | (1c) |
 | (2a) |
 | (2b) |
 | (2c) |
 | (2d) |
). In contrast, the best fit for the reduction of Y43L cyt c3 was obtained with Eq. 2d, and the curve obtained from the best-fit parameters is given in Fig. 4 B, and apparent rate constants are summarized in Table 4. Clearly, the analysis is limited by the ability to resolve individual rate constants and the use of models, which assume the minimum number of resolvable rate constants. Nevertheless, the data show a clear change in the rate constant for the fourth reduction step.
|
) was smaller than 0.030 Å for both structures. Eight and 17 ethanol molecules were identified in the wild-type and Y43L structures, respectively. In addition, two sulfate anions were clearly identified near Lys57 and Lys101.
The overall structures of the wild-type and Y43L cyt c3 are almost identical. The root mean square deviation for all identical protein atoms was found to be 0.525 Å between the wild-type and Y43L structures with the program SHELXPRO (Sheldrick and Schneider, 1997). The most striking feature of the Y43L mutant structure is the displacement of several residues in the N-terminal region in comparison with the wildtype (Fig. 5 A). The side-chain atom, C
1, of Leu43 pushes out the atoms of Ala0-Lys3 (Ala1-Lys4). The -carbon atom of Ala1 (Ala2) is displaced by 1.1 Å. In addition, the O
2 atom of Glu96 in the Y43L mutant was displaced by 1.1 Å. This residue is located in the vicinity of heme 2 but far from the mutated position. In the Y43L structure, the propionate groups at C-13 of heme 1 and heme 2 exist in two resolvable conformations. One of the carboxyl oxygens of the propionate group of heme 1 retains the hydrogen bond with the amide proton of Cys46 in both conformers as that found for the wildtype. The two conformers of heme 2 are shown in Fig. 5 B. The solvent accessibilities obtained by WHAT IF (Vriend, 1990) for hemes 1, 2, 3, and 4 of the wild type are 145, 161, 137, and 133 Å2, respectively. Those of the Y43L mutant are 142 (same for two conformers), 164 and 147 (for two conformers, respectively), 130, and 142 Å2, respectively.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
|---|
Although Leu and Phe are both hydrophobic, only Y43L substitution induced changes in the redox potentials. Therefore, the aromaticity should be responsible for lowering the redox potentials. The effect of the aromatic ring on the redox potentials was found local. Therefore, this should not be the major reason for the extremely low redox potentials of cyt c3. It is well-known that the bis-imidazole coordination and high exposure of the hemes are important factors to lower the redox potentials of cyt c3. The structural element mentioned above should be another complementary factor to lower them. While the aromatic ring orients parallel to the imidazole ring of His34 in the crystal structure of the oxidized form, it moves away from the ligand in the reduced solution structure (Harada et al., 2002). The 
- interaction between the stacked rings seems to stabilize the oxidized form of heme 1. Since the main chain of Y43 is located close to heme 2, the loss of the - interaction should also be associated with the large decrease in the interacting potential I12 for the Y43L mutant. This is consistent with the previous report (Harada et al., 2002) that Tyr43 is involved in the cooperative reduction between hemes 1 and 2. Furthermore, suppression of the intramolecular electron exchange rate was found for Y43L cyt c3, a situation not encountered with Y43F.
Laser flash photolysis of the Y43L protein provided an insight into the reduction mechanism. The apparent rate constant kiv was found to be 13x greater than ki, kii, and kiii, which were not resolvable, and 9x as large as kiv for the wild-type cyt c3 (Table 4). This means that the reduction rate of the three-electron-reduced form of Y43L cyt c3 is different from those of the other macroscopic oxidation forms and also from the same oxidation form of the wild type. The heme responsible for the major reduction of this oxidation form is heme 3 (Tables 2 and 3). However, the relatively small changes in redox potential induced by Y43L mutation do not provide a reasonable explanation for the significant change in kiv for Y43L relative to the other three rate constants and relative to kiv for the wild type. A significant change in individual rate constants could be ascribed to a significant change in heme accessibility. However, the crystal structures determined in this work do not indicate any large changes in heme accessibility in the fully oxidized state. In contrast, the Y43L mutation results in the slower intramolecular electron transfer, and it also appears to suppress the interaction between hemes 1 and 2. This can change the reduction rate of heme 3 through intramolecular electron transfer. Consequently, the 5-dRfH· reduction kinetics of the four hemes might not be independent processes, but instead correlated through intramolecular electron transfer.
|
ACKNOWLEDGEMENTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
|---|
This research was partly supported by grants from the Ministry of Education, Science, Technology, Sport and Culture of Japan (CREST to H.A.), and from the National Institutes of Health (GM21227 to M.A.C.).
|
FOOTNOTES |
|---|
Yoshiki Higuchi's current address is the Graduate School of Science, Himeji Institute of Technology, 3-2-1 Koto Kamigori, Hyogo 678-1297, Japan.
Abbreviations used: Tyr, tyrosine; Y43L, a mutation with Tyr43 replaced by leucine; NMR, nuclear magnetic resonance; 5-dRf, 5-deazariboflavin; DvMF, Desulfovibrio vulgaris Miyazaki F; DvH, D. vulgaris Hildenborough; Dg, D. gigas; DdA, D. desulfuricans ATCC 27774; DdE, D. desulfuricans Essex 6; Ds, D. salexigens; Dd, D. desulfuricans; Da, D. africanus; and Dmn, Desulfomicrobium norvegicum.
Submitted on June 22, 2003; accepted for publication July 23, 2003.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
|---|
Ambler, R. P. 1980. From Cyclotrons to Cytochromes. A. B. Robinson, and N. O. Kaplan, editors. Academic Press, London. 263–279.
Brünger, A. T. 1992. X-PLOR Vers. 3.1: A System for X-Ray Crystallography and NMR. Yale University Press, New Haven, CT.
Bruschi, M. 1994. Cytochrome c3 (Mr 26,000) isolated from sulfate-reducing bacteria and its relationships to other polyheme cytochromes from Desulfovibrio. Methods Enzymol. 243:140–155.[Medline]
Collaborative Computational Project, Number 4, SERC Darebury Laboratory. 1994. CCP4—a suite of programs for protein crystallography. Acta Cryst. D50:760–763.
Coutinho, I. B., and A. V. Xavier. 1994. Tetraheme cytochromes. Methods Enzymol. 243:119–140.[Medline]
Czjzek, M., F. Payan, and R. Haser. 1994. Molecular and structural basis of electron transfer in tetra- and octa-heme cytochromes. Biochimie. 76:546–553.[Medline]
Czjzek, M., F. Guerlesquin, M. Bruschi, and R. Haser. 1996. Crystal structure of a dimeric octaheme cytochrome c3 (M(r) 26,000) from Desulfovibrio desulfuricans norvegicum. Structure. 4:395–404.[Medline]
Czjzek, M., L. ElAntak, V. Zamboni, X. Molleri, A. Dolla, F. Guerlesquin, and M. Bruschi. 2002. Crystal structure of the hexadeca-heme cytochrome Hmc and structural model of its complex with cytochrome c3. Structure. 10:1677–1686.[Medline]
Dolla, A., L. Florens, P. Bianco, J. Haladjian, G. Voordouw, E. Forest, J. D. Wall, F. Guerlesquin, and M. Bruschi. 1994. Characterization and oxidoreduction of cytochrome c3 after heme axial ligand replacements. J. Biol. Chem. 269:6340–6346.
Fan, K., H. Akutsu, Y. Kyogoku, and K. Niki. 1990. Estimation of microscopic redox potentials of a tetraheme protein, cytochrome c3 of Desulfovibrio vulgaris Miyazaki F and partial assignments of heme groups. Biochemistry. 29:2257–2263.[Medline]
Harada, E., Y. Fukuoka, T. Ohmura, A. Fukunishi, G. Kawai, T. Fujiwara, and H. Akutsu. 2002. Redox-coupled conformational alterations in cytochrome c3 from D. vulgaris Miyazaki F on the basis of its reduced solution structure. J. Mol. Biol. 319:767–778.[Medline]
Hashimoto-Gotoh, T., T. Mizuno, Y. Ogasahara, and M. Nakagawa. 1995. An oligodeoxyribonucleotide-directed dual amber method for site-directed mutagenesis. Gene. 152:271–275.[Medline]
Higuchi, Y., M. Kusunoki, Y. Matsuura, N. Yasuoka, and M. Kakudo. 1984. Refined structure of cytochrome c3 at 1.8 Å resolution. J. Mol. Biol. 172:109–139.[Medline]
Kitamura, M., K. Ozawa, S. Kojima, I. Kumagai, H. Akutsu, and K. Miura. 1993. The primary structure of pre-cytochrome c3 from Desulfovibrio vulgaris (Miyazaki F) as determined by nucleotide sequencing of its gene and partial amino acid sequencing. Protein Seq. Data Anal. 5:193–196.
Leslie, A. G. W. 1990. MOSFILM Vers. 5.41, MRC Laboratory of Molecular Biology. In Crystallographic Computing. Oxford University Press, Oxford, UK.
Luzzati, V. 1952. Traitement statistique des erreurs dans la determination des structures cristallines. Acta Crystallogr. 5:802–810.
Mathews, F. S. 1985. The structure, function and evolution of cytochromes. Prog. Biophys. Mol. Biol. 45:1–56.[Medline]
Matias, P. M., A. V. Coelho, F. M. A. Valente, D. Plaido, J. LeGall, A. V. Xavier, I. A. C. Pereira, and M. A. Carrondo. 2002. Sulfate respiration in Desulfovibrio vulgaris Hildenborough: structure of the 16-heme Cytochrome c HmcA at 2.5 Å resolution and a view of its role in transmembrane electron transfer. J. Biol. Chem. 277:47907–47916.
Nørager, S., P. Legrand, L. Pieulle, C. Hatchikian, and M. Roth. 1999. Crystal structure of the oxidised and reduced acidic cytochrome c3 from Desulfovibrio africanus. J. Mol. Biol. 290:881–902.[Medline]
Niki, K., Y. Kobayashi, and H. Matsuda. 1984. Determination of macroscopic standard potentials of a molecule with a reversible n-consecutive one-electron transfer process. Application to a tetra-heme protein: cytochrome c3. J. Electroanal. Chem. 178:333–341.
Ohmura, T., T. Inobe, K. Kano, T. Horizumi, and H. Akutsu. 1997. Unusual behavior of a heme in a tetraheme protein, cytochrome c3 from Desulfovibrio vulgaris Miyazaki F, in the reduction process. J. Electroanal. Chem. 438:237–243.
Ozawa, K., A. I. Tsapin, K. H. Nealson, M. A. Cusanovich, and H. Akutsu. 2000. Expression of a tetraheme protein, Desulfovibrio vulgaris Miyazaki F cytochrome c3, in Shewanella oneidensis MR-1. Appl. Environ. Microbiol. 66:4168–4171.
Ozawa, K., F. Yasukawa, Y. Fujiwara, and H. Akutsu. 2001. A simple, rapid, highly efficient gene expression system for multiheme cytochrome c. Biosci. Biotechnol. Biochem. 65:185–189.[Medline]
Park, J.-S., T. Ohmura, K. Kano, T. Sagara, K. Niki, Y. Kyogoku, and H. Akutsu. 1996. Regulation of the redox order of four hemes by pH in cytochrome c3 from D. vulgaris Miyazaki F. Biochim. Biophys. Acta. 1293:45–54.[Medline]
Pollock, W. B. R., M. Loutfi, M. Bruschi, B. J. Rapp-Giles, J. D. Wall, and G. Voordouw. 1991. Cloning, sequencing, and expression of the gene encoding the high-molecular-weight cytochrome c from Desulfovibrio vulgaris Hildenborough. J. Bacteriol. 173:220–228.
Salgueiro, C. A., P. N. da Costa, D. L. Turner, A. C. Messias, W. M. A. M. van Dongen, L. M. Saraiva, and A. V. Xavier. 2001. Effect of hydrogen-bond networks in controlling reduction potentials in Desulfovibrio vulgaris (Hildenborough) cytochrome c3 probed by site-specific mutagenesis. Biochemistry. 40:9709–9716.[Medline]
Sheldrick, G. M., and T. R. Schneider. 1997. SHELXL: high-resolution refinement. Methods Enzymol. 277:319–343.[Medline]
Tollin, G., T. E. Meyer, and M. A. Cusanovich. 1986. Elucidation of the factors which determine reaction-rate constants and biological specificity for electron-transfer proteins. Biochim. Biophys. Acta. 853:29–41.[Medline]
Turner, D. L., C. A. Salgueiro, T. Catarino, J. LeGall, and A. V. Xavier. 1996. NMR studies of cooperativity in the tetraheme cytochrome c3 from Desulfovibrio vulgaris. Eur. J. Biochem. 241:723–731.[Medline]
Vriend, G. 1990. Quality control of protein models: directional atomic contact analysis. J. Mol. Graph. 8:52–56.[Medline]
Yagi, T., and K. Maruyama. 1971. Purification and properties of cytochrome c3 of Desulfovibrio vulgaris Miyazaki. Biochim. Biophys. Acta. 243:214–224.[Medline]
This article has been cited by other articles:
 |
|
 |
|
  S.-m. Kim, T. Yamamoto, Y. Todokoro, Y. Takayama, T. Fujiwara, J.-S. Park, and H. Akutsu Phospholipid-Dependent Regulation of Cytochrome c3-Mediated Electron Transport across Membranes Biophys. J., January 15, 2006; 90(2): 506 - 513. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
at the ith reduction step) and microscopic (
and 
respectively) redox potentials, and the interacting potentials Iij
