Role of tryptophan 121 in the soluble CuA domain of cytochrome c oxidase: structure and electron transfer studies

Ai-Xin Song1, Lian-Zhi Li1, Ting Yu1, Shi-Ming Chen2 and Zhong-Xian Huang1,3

1Chemical Biology Laboratory, Department of Chemistry and 2Research Center for Analysis and Measurement, Fudan University, Shanghai 200433, China

3 To whom correspondence should be addressed. e-mail: zxhuang{at}fudan.edu.cn


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
To investigate the contribution of tryptophan-121 (Trp121) residue to the structure and function of soluble CuA domain of cytochrome c oxidase, three mutant proteins, Trp121Tyr, Trp121Leu and Trp121-deleted mutant of the soluble domain of Paracoccus versutus cytochrome c oxidase, were constructed and expressed in Escherichia coli BL21 (DE3). Optical spectral studies showed that both the coordination structure of the CuA center and the secondary structure of the protein were changed significantly in the Leu substitution and deletion mutants of Trp121. Their electron transfer activity with cytochrome c was inhibited severely, as shown in stopped-flow kinetic studies. However, the CuA center can be reconstructed in the Trp121Tyr mutant although its stability decreases compared with the wild-type protein. This mutant keeps the same secondary structure as the wild-type protein, but can only transfer electrons with cytochrome c at a rate of one-seventh-fold. Based on the information on the structure, we also investigated and analyzed the possible factors that affect electron transfer. It appears that the aromatic ring, the size of the side chain and the hydrogen bonding ability of the Trp121 are crucial to the structure and function of the soluble CuA domain.

Keywords: electron transfer/site-directed mutagenesis/soluble CuA domain/structure transition


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cytochrome c oxidase (CcO) is the terminal enzyme of the redox respiratory chain in mitochondria and many aerobic bacteria. It catalyzes the reduction of oxygen to water by accepting an electron from cytochrome c (cyt c) and then transferring it to oxygen molecules (Babcock and Wikström, 1992Go). Electron transfer (ET) between redox proteins has been an interesting subject in recent years. Many studies with cyt c and its different redox partners have demonstrated that efficient ET depends on interactions between proteins. The electrostatic interactions mediated by lysines on cyt c and acidic residues on the counterpart guide their encounter and binding and the hydrophobic interactions contribute significantly to the configuration optimal for ET (Williams et al., 1995Go; Witt et al., 1998aGo; Sun et al., 1999Go).

For the system of CcO and cyt c, several different methods, including chemical modification, cross-linking, monoclonal antibodies (Bisson et al., 1982Go; Millett et al., 1983Go; Taha and Ferguson-Miller, 1992Go) and site-directed mutagenesis (Witt et al., 1998aGo; Wang et al., 1999Go; Zhen et al., 1999Go), have proved that the soluble domain of subunit II (sdII) of CcO is its primary binding part with cyt c. The surface acidic residues (Gln120, Glu126, Asp159 and Asp178) and the hydrophobic residues (Trp121, Tyr122, Ile117 and Leu137) of sdII contribute to the interactions between proteins. Among these residues, Trp121 has been suggested to be the electron entry site to CcO (Witt et al., 1998bGo). The mutation to this residue (W121Q) almost inhibited completely the ET between CcO (from Paracoccus denitrificans) and cyt c although the binding was not obviously affected. Another similar result has also been reported in the study of the CcO enzyme of Rhodobacter sphaeroides, where the ET activity of the W121A and W121F (W121 corresponds to W143 in R.sphaeroides) mutants with cyt c has been decreased to 1 and 2%, respectively (Zhen et al., 1999Go).

The drastic inhibition of mutation at Trp121 to ET is surprising and the chemical mechanism is unknown. At present, no detailed information has been provided about how the Trp121 mediates the ET. It seems that the structural complexity of the whole oxidase makes further investigation difficult.

Since the cyt c interacts with CcO mainly through its soluble domain of subunit II, the study of an isolated sdII would potentially provide an understanding of the structure–activity relationships. At the same time, the simpler structure of sdII compared with CcO whole enzyme is also very helpful. X-ray investigation (Iwata et al., 1995Go; Tsukihara et al., 1995Go) shows that the soluble domain of subunit II in CcO has a characteristic ß-sheet structure. Ten ß-strands forms a ß-barrel fold; further, a C-terminal {alpha}-helix and a 310-helix are included (Figure 1A). Its active center, a special binuclear mixed-valenced purple CuA center, has attracted much interest because of its unusual spectroscopic properties (Malmström and Aasa, 1993Go; Palmer, 1993Go; Beinert, 1997Go) and because it is believed to be the first active metal site of electron entry into the oxidase (Hill, 1994Go; Adelroth et al., 1995Go; Ferguson-Miller and Babcock, 1996Go; Malatesta et al., 1998Go). The hydrophobic residue Trp121 is located at the surface of subunit II ~5 Å above the CuA center (Figure 1B) and is a member of the highly conserved aromatic residues cluster (Trp121, Tyr122, Trp123, Tyr125 and Tyr127). The special location and high conservation of Trp121 suggest its importance to the structure and function of the protein.



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Fig. 1. (A) Schematic structure of the soluble domain of subunit II and (B) the coordination geometry of the CuA center. The coordinates are from the P.denitrificans structure (1ar1.pdb).

 
In this paper, we report the effect of mutation at Trp121 on the soluble CuA domain for the first time. The wild-type of the soluble CuA domain of Paracoccus versutus and its three mutants W121Y, {Delta}W121 and W121L were expressed and purified from Escherichia coli. Using spectral and kinetic techniques, we studied and compared the structure, property changes and ET function of these mutants. The results show that different mutations at Trp121 have very different effects on the CuA center and protein structure and also ET function. The transitions in copper coordination geometry and protein conformation were observed in the soluble domain. A possible mechanism of Trp121 in facilitating ET reaction is also suggested.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials

Horse heart cytochrome c (type VI) was purchased from Sigma. Reduced cyt c was prepared by adding ascorbic acid to cyt c solution and separated with a Sephadex G-25 column just before use. The cyt c concentration was determined from its absorbance at 550 nm ({epsilon}550 = 29.5 mM–1 cm–1). The pET11d plasmid containing sdII gene, which encodes for amino acid residues 100–252 of P.versutus CcO subunit II, was a kind gift from Professor G.W.Canters of Leiden University, The Netherlands. Bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris, ultra-pure) was obtained from Amresco. All other chemicals were of reagent grade.

Preparation of the wild-type and the mutant proteins of sdII

Site-directed mutagenesis was performed according to the overlap extension polymerase chain reaction (PCR) method (Higuchi et al., 1988Go). The codon of Trp (TGG) of sdII was mutated to TAT (Tyr) or CTG (Leu) or deleted. The DNA constructs were confirmed by the dideoxyribonucleotide method using an ABI PRIME 3700 automated sequencer.

The expression and purification of the wild-type and mutant CuA domain were generally performed according to the literature (Lappalainen et al., 1993Go). Some variations were as follows. The E.coli cells were broken with sonication and lysozyme. Almost the entire subunit II fragments were in inclusion bodies. These pellets were further dissolved in 6 M urea and the protein was refolded by the dilution method in the presence of dithiothreitol (DTT). After this, Cu+/Cu2+ was added to the protein solution by two-step dialysis, then the recombinant protein supernatant was purified through a Mono Q HR 10/10 column on a Pharmacia FPLC instrument. The purified protein was lyophilized to powder and kept at –20°C.

Spectroscopic studies

Electrospray mass spectra were recorded with an Esquire 3000 ion trap mass spectrometer (Bruker Daltonics, Germany). UV–visible spectra were recorded on a Hewlett-Packard Model 8453 diode-array spectrophotometer. Circular dichroism (CD) spectra were measured on a Jasco 715 spectropolarimeter with a 0.1 cm cell. Electron paramagnetic resonance (EPR) measurements were performed on a Bruker ER200D-SRC X-band spectrometer and the data were analyzed with the program ASPECT 3000.

The thermal stability of the CuA center structure was estimated by monitoring the change in absorption near 478 nm using the HP8453 spectrophotometer. The spectrophotometer was equipped with a Neslab RTE-5B circulating bath system that permitted the sample temperature to be stepped from 20 to 90°C (±0.2°C). The initial absorbance of protein was adjusted to 0.6–0.7 in 20 mM Bis-Tris buffer (pH 7.0) and paraffin oil was added to the surface of the protein solution before heating.

Rapid kinetic measurements

Rapid kinetic experiments were conducted on an SF-61DX2 double-mixing stopped-flow apparatus (Hi-Tech, UK) equipped with a Neslab RTE-5B circulating bath system. The reactions between cyt c and the CuA domain were monitored at a wavelength of 550 nm, which is the characteristic peak of reduced cyt c. Measurements for all of the proteins were carried out at 20 ± 0.2°C in 20 mM Bis-Tris (pH 7.0) containing 25 mM KCl. Over the course of the reaction 512 data points were recorded in each experiment and the average of at least 10 independent measurements was fitted to a single-exponential procedure (Specifit program). The observed rate constant of the reaction was plotted against the cyt c concentration and the bimolecular rate constant was calculated from the slope.

Miscellaneous

SDS–PAGE was performed in 15% gel (Laemmli, 1970Go). The concentration of the protein was determined by Bradford method using Coomassie Brilliant Blue G-250 (Bradford, 1976Go). The estimated molar extinction coefficients ({epsilon}278) for the wild-type and mutants are 52.17, 43.84, 37.68 and 37.09 mM–1 cm–1, respectively. Potential measurement of the CuA domain was carried out by the method of equilibrium titration and the change in the wavelength of 550 nm of reduced cyt c was monitored as an index of the redox process.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Preparation of the wild-type and mutants of CcO sdII

Based on DNA sequence analysis, all of the mutant genes were constructed correctly. We expressed and purified successfully the wild-type and the mutants of the soluble domain and the SDS–PAGE analysis for purified protein is shown as supplementary data [figure 1 (available at http://protein.oupjournals.org/)]. Electrospray mass spectrometric results for the wild-type protein and the W121Y, {Delta}W121 and W121L mutants of CcO sdII indicate that the molecular weights of the proteins are 17 117.10 ± 3.26, 17 097.00 ± 1.88, 16 930.94 ± 3.67 and 17 046.03 ± 1.80 Da, respectively. These values agree well with the molecular weights calculated from amino acid compositions of apo-sdII (17 119, 17 097, 16 934 and 17 047 Da, respectively), which further confirm completely successful mutagenesis.

After refolding and metal reconstitution, the W121Y mutant shows the characteristic purple color of the CuA center similar to the wild-type protein (Malmström and Aasa, 1993Go; Palmer, 1993Go; Beinert, 1997Go), but the W121L mutant and most of the {Delta}W121 mutant are colorless. Furthermore, the elute behaviors of the W121L and {Delta}W121 mutants from the Mono Q column are also different from that of the wild-type protein [supplementary data, figure 2 (available at http://protein.oupjournals.org/)]. The increase of binding strength on Mono Q column suggests the structure alternation or residue rearrangement in these two mutant proteins; however, the {Delta}W121 mutant does not behave entirely as the W121L mutant. A small part (7%) of the {Delta}W121 could be properly reconstituted into the CuA center (so-called purple {Delta}W121 mutant) and was eluted from the Mono Q column at the same salt concentration as the wild-type sdII [supplementary data, figure 2 (available at http://protein.oupjournals.org/)].

Spectroscopic studies: the structure and properties of sdII and its mutant proteins

The UV–visible absorption spectra of the wild-type and the three mutant proteins of CcO sdII are shown in Figure 2. In the wavelength range 320–900 nm, the absorption peaks of the wild-type protein at 365, 478, 530 and 810 nm feature a typical CuA center (Gamelin et al., 1998Go). These characteristics are similar to that of the CuA center from P.denitrificans and also other native and engineered CuA centers (Table I). The UV–visible spectrum of the W121Y mutant is similar to that of the wild-type protein except for a slight shift of the absorption peaks (478 -> 474 and 810 -> 800 nm). For the W121L and {Delta}W121 mutants, the above character diminished, which suggested that the CuA coordination structure was not formed.



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Fig. 2. UV–visible absorption spectra of the wild-type and mutant proteins. The samples were dissolved in 20 mM Bis-Tris buffer, pH 7.0. WT, solid line; W121Y, dashed line; {Delta}W121, dotted line; W121L, dash-dotted line; purple {Delta}W121, dash-dot-dotted line.

 

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Table I. UV–visible absorption and EPR spectroscopy data for different CuA centers
 
Figure 2 also shows the absorption spectrum of the purple {Delta}W121 mutant. The typical CuA feature is obvious except for the much greater blue shift in the near-IR region (810 -> 770 nm). The CuA center stability of the purple {Delta}W121 mutant decreases significantly. The characteristic purple color of the CuA center diminished ~60 h after elution from the column, whereas the color showed no significant change for the wild-type protein and the W121Y mutant during the same period. The blue shift in near-IR region absorption manifests the reduction of distance between the Cu–Cu atoms (Hay et al., 1998Go), and this may explain the decrease in protein stability.

Figure 3 shows the CD spectra of the wild-type and mutant proteins of the soluble domain. The far-UV CD spectra of the wild-type and the W121Y variant have a negative peak at 215 nm, which demonstrates a typical ß-sheet structure (Slutter et al., 1996Go). This feature is in accordance with the structure of sdII reported in the whole oxidase. In contrast, the CD characteristics of the {Delta}W121 and W121L mutants are double negative peaks at 208 and 220 nm. This transition from a single- to a double-negative peak shows the relative decrease of ß-sheets (or the relative increase of helix abundance) in the {Delta}W121 and W121L mutants (Berova et al., 2000Go). The near-UV CD spectra of the mutants also show obvious changes with respect to the wild-type protein, signifying the rearrangement in local tertiary structure of the protein (Berova et al., 2000Go).



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Fig. 3. Far-UV (top) and near-UV (bottom) CD spectra of the wild-type and mutant proteins. The concentration of proteins are 10–14 µM.

 
The structure conversion of the proteins is accompanied by a coordination change of the CuA center. As corroborated by the EPR results shown in Figure 4 and Table I, the signal of the wild-type protein clearly arises from the CuA center (Farrar et al., 1995Go; Beinert, 1997Go), its g// and g{perp} being 2.17 and 2.06, respectively. The W121L and {Delta}W121 mutant proteins exhibit a copper EPR spectrum that is dominated by features of a type II Cu2+ species (Nilsson et al., 1988Go; Wittung-Seafshede et al., 1998Go; Yuan et al., 1998Go) and g// is 2.29 and A// is ~167 G. For the W121Y mutant protein, the CuA feature is still obvious but disturbed to some extent by the signal of the type II Cu2+.



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Fig. 4. EPR spectra recorded at 100 K. (a) WT (0.87 mM); (b) W121Y (0.67 mM); (c) W121L (0.40 mM); (d) {Delta}W121 (0.55 mM). The samples were in 30% glycol; microwave frequency, 9.68 GHz; microwave power, 10 mW; modulation amplitude, 5 G.

 
In the three mutants, the W121Y mutant is the only one that can reconstitute the CuA center. We further compared the thermal stability of the CuA center in the W121Y mutant with that of the wild-type sdII. As presented in Figure 5, the stability of the CuA center in the W121Y mutant is lower than that of the wild-type protein. The transition temperature (Tm) for the CuA center is ~56.6 ± 0.2°C for the wild-type protein and ~53.4 ± 0.2°C for the W121Y mutant. The decrease in Tm illustrates that in the W121Y mutant protein the replacement of Trp121 has disturbed the CuA center.



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Fig. 5. The thermal stability of the CuA center. Circles, absorption change of WT at 478 nm; squares, change of W121Y at 474 nm.

 
Effect of mutation at Trp121 on electron transfer with cytochrome c

The ET reactions of sdII (the wild-type or its variants) with horse heart cyt c were studied under the same conditions. For the wild-type and W121Y proteins, the absorbance changes can be fitted to a single-exponential decay (Figure 6), indicating the presence of only one kinetically active cyt c binding site (Lappalainen et al., 1995Go). However, the situations are very different for the {Delta}W121 and W121L mutant proteins. The absorbance of 550 nm did not change under the above conditions (Figure 6), which suggested that no obvious electron transfer reaction took place between these two mutants and cyt c. The observed rate constants (kobs) of the wild-type and the W121Y mutant protein reacting with cyt c were plotted against the cyt c concentration (Figure 7) and the bimolecular rate constants (k12) were calculated, and were 2.88x105 M–1 s–1 (WT) and 0.40x105 M–1 s–1 (W121Y). The k12 value of the wild-type protein is very close to the value (3.00x105 M–1 S–1) of the soluble domain from P.denitrificans (Lappalainen et al., 1995Go).



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Fig. 6. Kinetic traces for ET from reduced cytochrome c to the soluble domain (WT and mutants) monitored at 550 nm. The reaction was measured in a solution of 20 mM Bis-Tris, 25 mM KCl, pH 7.0 (20°C). The concentration of the wild-type and W121Y mutant proteins are 4 µM and the concentration of cyt c is 30–35 µM.

 


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Fig. 7. Second-order plots of cytochrome c oxidation by the wild-type (squares) and W121Y (circles) proteins. The rate constants k12 were obtained from the slopes.

 
The relative redox potential of the proteins can be considered as a measurement of the driving force ({Delta}G) in ET reaction. The results from the equilibrium titration experiment show that the relative redox potential of the wild-type CuA center is ~250 ± 3 mV, which is identical with the value reported in the literature (Lappalainen et al., 1993Go, 1995). The redox potential of the W121Y mutant is 242 ± 5 mV under the same experimental conditions. The nearly equal potentials of these two proteins show that the {Delta}G for ET is similar.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Site-directed mutagenesis is a powerful method for studying the contribution of a particular amino acid to the structure and function of proteins. In our work, Trp121 was mutated to Tyr or Leu or deleted. Tyrosine, as a conservative substitution, retains aromaticity and the ability to form hydrogen bonds. Leucine is an aliphatic hydrophobic residue. With these two mutations, we tried to understand the effect of aromaticity and hydrophobicity on this site (Overholtzer et al., 1996Go; Witt et al., 1998bGo). Concerning the design of the deletion mutant, {Delta}W121, our intention is to study whether the adjacent Tyr122 residue (another residue of an aromatic cluster) will take over the function of the Trp121 in the structure and ET as suggested in a previous paper (Witt et al., 1998bGo).

The isolated wild-type soluble domain from P.versutus in this work has the characteristic CuA center and secondary structure just as that in the CcO whole enzyme. Our mutation studies show that Trp121 plays important roles in maintaining the CuA active center, the backbone structure of sdII and its ET activity.

Trp121, CuA center and the structure of sdII

The CuA center and the protein structure of the soluble domain were sensitive to changes of the Trp121 residue. For the W121L and most of the {Delta}W121 mutant proteins, transitions in copper coordination geometry and protein conformation were observed. We deduce that these effects should have a close relationship with the special location of Trp121 in the protein. The crystal structures of CcO (see Figure 1B) show that Trp121 can form a hydrogen bond with Cys220 and His181 and contacts with Met227 via van der Waals force (Iwata et al., 1995Go). Because these three residues are the ligands of the CuA center, the stabilization of Trp121 to the CuA center is reasonable. The mutation at Trp121 will destabilize the CuA center, change the coordination structure and lead to a conformation transition of the protein.

However, a significant transition did not occur in the substitution of Trp for Tyr. The W121Y mutant has a similar CuA center and secondary structure to the wild-type sdII, except for some subtle shifts in the absorption peak maxima in the UV–visible spectra and a slight decrease in thermal stability. These facts show that replacement with an amino acid that has different properties can lead to different results. Compared with a Leu residue, Tyr has an aromatic group just as a Trp residue, so the mutation results suggest the significance of the aromatic ring at this site. The low hydrophobicity of the Leu residue is insufficient to maintain the integrity of the CuA center in the soluble domain.

In addition, the occurrence of the purple {Delta}W121 mutant during the preparation demonstrates that the protein still has the ability to form a CuA center and native structure when Trp121 is deleted. Since the CuA center can also be reconstituted in the W121Y mutant, we deduce that the formation of the purple {Delta}W121 mutant must depend on a certain aromatic residue, Tyr122, that is, the adjacent Tyr122 would partially take over the role in stabilizing the CuA center. Nevertheless, the increasing steric constraints due to the deletion of the residue destroy this compensation from Tyr122 for the deletion of Trp121 and finally lead to conformation transformation.

Possible mediating mechanism of the Trp121 on ET

Kinetic experiments in the soluble domain provide evidence for the importance of Trp121 to ET. No ET reaction is detected when the {Delta}W121 and W121L mutant is mixed with cyt c under the same conditions as the wild-type protein. Combined with the structural information, we ascribe this inactivity to the disruption of the CuA center. In contrast, the W121Y mutant did react with cyt c although the electron transfer rate decreased 7-fold. So far, this mutant retains the highest reactivity of all of the reported Trp121 mutants.

The reservation of the CuA center is no doubt the basis of the reactivity of the W121Y mutant protein. However, the CuA center alone cannot ensure the high ET activity. The existence of a hydroxyl group is also essential to the ET activity. As we mentioned in the Introduction, when the Trp121 residue was mutated into Phe121 in CcO of R.sphaeroides, which is also a CuA-containing variant [W121 corresponds to W143 in R.sphaeroides (Zhen et al., 1999Go)], but with an ET activity with cyt c far lower than that of the W121Y mutant protein, only 2% of the activity of the wild-type protein remains. This result also shows that the sole aromatic ring of Phe cannot carry out the ET function. The different effect of the W121Y and W121F mutants on the ET rate could be ascribed to the difference between Tyr and Phe residues. Because these two residues have almost the same sized side chain (Caffrey and Cusanovich, 1994Go), the hydroxyl group of Tyr, having the ability to form hydrogen bonds just like the nitrogen of the Trp residue, should play an important role in the ET reaction. The X-ray structure of CcO showed that there was actually a hydrogen bond between Trp121 and Cys220 (Iwata et al., 1995Go) and Medvedev et al. proved theoretically that this hydrogen bond was responsible for effective ET via the Cys220 pathway (Medvedev et al., 2000Go). Here, we provide solid evidence for the necessity for hydrogen bonding in the ET reaction.

Since the residue 121 is not involved in the direct electrostatic binding between CcO and cyt c (Witt et al., 1998Go; Zhen et al., 1999Go) and the driving force ({Delta}G) for the ET reaction is essentially unchanged for the W121Y mutant compared with the wild-type protein, then what are the possible factors that cause the 7-fold reduction in the ET rate of the W121Y mutant?

The effect of mutation on the ET pathway is a considerable factor. According to the interaction model proposed by Roberts and Pique, a through-space gap may exist between the edge of the heme c and the Trp121 residue (Roberts and Pique, 1999Go; Wang et al., 1999Go). The electron will jump through this gap in the reaction, so the smaller side-chain size of 34 Å3 in the tyrosine residue (Caffrey and Cusanovich, 1994Go) must make an effective ET reaction more difficult.

Furthermore, the stability of the active center in the protein is also important to its function. Since the Tm value of the CuA center in the W121Y mutant is lower than that in the wild-type protein, we predicted that the decreased stability of the CuA center in the W121Y mutant might be another important factor affecting ET.

In summary, our results clearly demonstrate that Trp121 is crucial to the CuA center, backbone conformation and ET activity of the soluble domain. The structural transition is an important factor causing changes in ET function. The aromatic ring of Trp121 and its hydrogen bonding interaction with the copper ligands stabilize the CuA center structure and facilitate ET, and the size of the side chain is directly responsible for the efficiency of ET. Residue substitution at this site will perturb or disrupt the local environment, consequently bringing about substantial changes in structure, properties and function. Finally, we would mention that the structure transition due to the mutation of Trp121 has not been reported previously in mutants of whole oxidase. Hence, studies on the soluble CuA domain provide a new insight into the role of Trp121 in proteins and the ET pathway.


    Acknowledgements
 
We gratefully acknowledge Professor G.W.Canters of Leiden University, The Netherlands, for his kind gift of the cytochrome c oxidase soluble domain gene. This study was supported by the National Natural Science foundation of China.


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 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received September 11, 2002; revised February 26, 2003; accepted May 27, 2003.





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