Analysis of crystal structure of the Phe35fiTyr mutant shows that the overall structure of the mutant is basically the same as that of the wild-type protein.. However, the intro-duction o
Trang 1X-ray crystallography, CD and kinetic studies revealed the essence
Ping Yao1, Jian Wu2, Yun-Hua Wang1, Bing-Yun Sun1, Zong-Xiang Xia2and Zhong-Xian Huang1
1
Chemical Biology Laboratory, Department of Chemistry, Fudan University, Shanghai, People’s Republic of China;
2
State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry,
Chinese Academy of Sciences, Shanghai, People’s Republic of China
Conserved phenylalanine 35 is one of the hydrophobic patch
residues on the surface of cytochrome b5(cyt b5) This patch
is partially exposed on the surface of cyt b5while its buried
face is in direct van der Waals’contact with heme b
Resi-dues Phe35 and Phe/Tyr74 also form an aromatic channel
with His39, which is one of the axial ligands of heme b By
site-directed mutagenesis we have produced three mutants of
cyt b5: Phe35fiTyr, Phe35fiLeu, and Phe35fiHis We
found that of these three mutants, the Phe35fiTyr mutant
displays abnormal properties The redox potential of the
Phe35fiTyr mutant is 66 mV more negative than that of the
wild-type cyt b5 and the oxidized Phe35fiTyr mutant is
more stable towards thermal and chemical denaturation
than wild-type cyt b5 In this study we studied the most
interesting mutant, Phe35fiTyr, by X-ray crystallography,
thermal denaturation, CD and kinetic studies of heme
dissociation to explore the origin of its unusual behaviors Analysis of crystal structure of the Phe35fiTyr mutant shows that the overall structure of the mutant is basically the same as that of the wild-type protein However, the intro-duction of a hydroxyl group in the heme pocket, and the increased van der Waals’and electrostatic interactions between the side chain of Tyr35 and the heme probably result in enhancement of stability of the Phe35fiTyr mutant The kinetic difference of the heme trapped by the heme pocket also supports this conclusion The detailed confor-mational changes of the proteins in response to heat have been studied by CD for the first time, revealing the existence
of the folding intermediate
Keywords: cytochrome b5; folding; mutagenesis; stability; structure
Cytochrome b5(cyt b5) is a membrane-bound hemoprotein
It consists of a water-soluble, heme-containing domain and
a short hydrophobic tail of approximate 40 amino acid
residues that anchors the protein to the microsomal
membrane [1] The water-soluble domain functions as an
electron mediator in the cytochrome P450 reductase system
[2] and in the fatty acid desaturation system [3], etc In
erythrocytes, cyt b5 also exists as a soluble heme-binding
protein lacking the hydrophobic tail where its physiological
role is to reduce methemoglobin [4]
On the surface of cyt b5, there is a cluster of negatively
charged residues surrounding the exposed heme edge These
acidic residues have been proved to bind to the basic residues of the protein redox partners, such as cytochrome c [5,6], cytochrome P450 [7], metmyoglobin [8] and methe-moglobin [9] On the surface of cyt b5, there is also a hydrophobic patch of 350 A˚2 that is surrounded by negatively charged residues [10] The patch consists of the hydrophobic residues, Phe35, Pro40, Leu70 and Phe/Tyr74 and is totally conserved among different species This patch
is partially exposed to the surface of cyt b5, while its buried part is in direct van der Waals’contact with the heme [11] Residues Phe35 and Phe/Tyr74 also form an aromatic channel with His39, which is one of the axial ligands of heme b In addition, it has been reported that Phe35 as well
as Phe58 stabilizes the heme binding through aromatic interactions with the heme ring system [12]
To illustrate the possible roles of the negative patch as well as the aromatic channel, we previously designed and constructed three Phe35 mutants of cyt b5, Phe35fiTyr, Phe35fiLeu, and Phe35fiHis [13] In that study we found that of the three mutants, the Phe35fiTyr mutant displayed abnormal properties The redox potential of the Phe35fi Tyr mutant is 66 mV more negative than that of the wild-type cyt b5 [14], and the oxidized Phe35fiTyr mutant is obviously more stable towards heat and chemical denatur-ation than wild-type cyt b5 [13] We also studied electron transfer reactions of cyt b5 Phe35fiTyr and Phe35fiLeu variants with cytochrome c, with the wild-type and the Tyr83Phe, Tyr83Leu variants of plastocyanin, and with the inorganic complexes [Fe(EDTA)]–, [Fe(CDTA)]– and [Ru(NH3)6]3+ The change at Phe35 of cyt b5did not affect the second-order rate constants of the electron transfer
Correspondence to Z.-X Huang, Chemical Biology Laboratory,
Department of Chemistry, Fudan University, Shanghai 200433,
China Fax: + 86 21 65641740, Tel.: + 86 21 65643973,
E-mail: zxhuang@fudan.edu.cn
Z.-X Xia, State Key Laboratory of Bio-organic and Natural Products
Chemistry, Shanghai Institute of Organic Chemistry,
Chinese Academy of Sciences, Shanghai 200032, China.
Fax: +86 21 64166128, Tel.: + 86 21 64163300,
E-mail: xiazx@pub.sioc.ac.cn
Abbreviations: cyt b 5 : cytochrome b 5 ; Tb 5 : trypsin-solubilized bovine
liver microsomal cytochrome b 5 ; Lb 5 : lipase-solubilized bovine liver
microsomal cytochrome b 5 ; Mb: myoglobin; r.m.s., root mean square.
Note: P Yao and J Wu made equal contributions to this work.
Note: the atomic coordinates have been deposited in Protein Data
Bank: PDB ID 1M20.
(Received 16 January 2002, revised 2 July 2002,
accepted 17 July 2002)
Trang 2reactions These results show that the invariant aromatic
residues and aromatic channel are not essential for electron
transfer in these systems [15]
Because mutation at Phe35 causes changes in functional
properties, and site-directed mutations rarely leads to
increasing stability, it would be most interesting to reveal
the essential difference between the wild-type and mutant
proteins and to give a proper interpretation In this paper,
the secondary structural changes of cyt b5and its Phe35fi
Tyr mutant towards heat have been characterized by CD
Meanwhile, the heme dissociation and transfer reactions also
provide a good means of examining the subtle local
conformation changes around the heme group under natural
conditions Therefore, the heme dissociation kinetics at
different urea concentrations and the heme transfer reactions
between the wild-type cyt b5or its Phe35fiTyr mutant and
apo-myoglobin (Mb) were studied to demonstrate the
affinity changes of the heme with cyt b5polypeptide chain
In this paper the crystal structure of the cyt b5Phe35fiTyr
mutant has been determined by X-ray analysis Based on the
molecular structure and the above detailed studies the
essence of these unusual behaviors is discussed
M A T E R I A L S A N D M E T H O D S
Protein preparation
Bovine liver cyt b5 and its mutants were prepared and
purified as described previously [13] The concentrations of
ferricytochrome b5and the mutants were determined with
the value of OD414¼ 117 mM )1Æcm)1[16] Horse skeletal
Mb was from Sigma and was purified according to the
method described by Hagler et al [17] Apo-Mb was
prepared according to the method of La Mar et al [18]
The concentrations of Mb and apo-Mb were determined
with the values of e409¼ 171 mM )1Æcm)1 [19], and
e280¼ 15.2 mM )1Æcm)1[20], respectively
X-ray analysis of cytochromeb5Phe35fiTyr mutant
Crystallization Single crystals of the Phe35fiTyr mutant
of trypsin-solubilized bovine liver microsomal cytochrome
b5 (Tb5) were grown by the vapor diffusion method in
hanging drops containing 10 mgÆmL)1protein solution in
3.1–3.2M phosphate buffer (pH 7.5) at 20C This is
similar to the crystallizing condition used for wild-type Tb5
[21] and lipase-solubilized bovine liver microsomal
cyto-chrome b5(Lb5) [22] The typical size of the single crystals
was 0.6 · 0.5 · 0.3 mm Crystals of wild-type Tb5 [21]
and the Tb5 Val61fiHis mutant [23] are isomorphous
belonging to the monoclinic space group C2 with the
following unit cell parameters: a¼ 70.71 A˚, b ¼ 40.39 A˚,
c¼ 39.30 A˚ and b ¼ 111.72
The X-ray diffraction data of the Phe35fiTyr mutant
were collected up to 1.8 A˚ resolution using one single crystal
on the MarResearch Imaging Plate-300 Detector System at
room temperature Data processing was accomplished with
the programsDENZOandSCALEPACK[24], giving an Rsymof
6.1% and data completeness of 94.3% The crystal data and
the data collection statistics are summarized in Table 1
Structure solution and crystallographic refinement The
structure determination and refinement of the cyt b
Phe35fiTyr mutant were carried out using the program packagesX-PLOR[25] andCNS[26] successively on a Silicon Graphics Indigo 2 workstation All the data up to 1.8 A˚ were used for structural refinement at theCNSrefinement stage A random sample of 10% of the X-ray data was excluded from the refinement and was taken as the test data set, and the agreement between the calculated and observed structure factors of the test data set was monitored throughout the course of the refinement The graphics software TURBO-FRODO [27] was used for the model rebuilding
The initial structural model of the Phe35fiTyr mutant was determined using the difference Fourier method based
on the crystal structure of the Val61His mutant of cyt b5at 2.1 A˚ resolution [23], from which all of the solvent molecules and the side chain of His61 were omitted Rigid body refinement, limited to 2.2 A˚ resolution, yielded an R factor of 27.1% and an Rfree of 28.4% The positional refinement and temperature factor refinement were carried out for each round using the programX-PLOR The program
TURBO-FRODOwas used to fit the side chains of Tyr35 and Val61, and then the model was adjusted manually to improve the fitting of the model by using the (2Fo-Fc) and the (Fo-Fc) electron density maps calculated regularly during the refinement When the resolution was gradually extended to 2.0 A˚, the solvent molecules were fitted to the peaks higher than 3 r in the (Fo-Fc) electron density map if the sites satisfied reasonable distance and geometry criteria Those water molecules without a reasonable hydrogen-bonding environment and with a thermal factor > 50 A˚2 were removed from the final model
The structure was further refined by using the more powerful program packageCNS The simulated annealing refinement starting from 2500 K with a cooling rate of 25 K per cycle was carried out, followed by the individual temperature factor refinement
Thermal denaturation of cytb5monitored by CD
CD spectra of cyt b5and its variants were recorded with a Jasco J-715 spectropolarimeter equipped with a Naslab temperature controller The path length was 0.1 cm in the 190–250 nm region and 1 cm in the 250–500 nm region,
Table 1 Crystal data and data collection statistics.
Cell dimensions
h I/r(I) i c
23.5 (3.9)b a
R sym ¼ SUM (ABS (I- h I i))/SUM (I) b The numbers in the parentheses correspond to the data in the highest resolution shell (1.80–1.84 A˚).cMean signal-to-noise ratio.
Trang 3respectively The ellipticity was recorded at 100 nmÆmin)1
speed, 0.2 nm resolution, five accumulations, 1.0 nm
bandwidth Cyt b5 or its mutant was dissolved in the
phosphate buffer (100 mMpH 7.0) The protein
concentra-tions were 25 lMin the 190–250 nm region and 12.5 lMin
the 250–500 nm region, respectively At each given
tem-perature, the protein sample was allowed to equilibrate for
20 min before the spectrum was recorded The temperature
was increased stepwise over the range 30–95C and the
temperature accuracy was within ± 0.1C
Urea- and guanidine hydrochloride-mediated
denaturation of cytb5variants
For the kinetic study of urea-mediated denaturation of
cyt b5and its variants, the time course of the absorbance
increase at 412 nm was recorded immediately after mixing
of 0.3 mL cyt b5and 2.7 mL urea or guanidine
hydrochlo-ride at 30C The protein solution was prepared in a
100 mMphosphate buffer (pH 7.0) The final concentration
of cyt b5 was 4 lM, and the concentration of urea and
guanidine hydrochloride varied from 0 to 10 and from 0 to
6M, respectively All measurements were carried out on a
HP 8452A diode-array spectrophotometer
(Hewlett-Pac-kard) The kinetics of heme dissociation from cyt b5by urea
was analyzed as described in the literature [28,29]
Heme-transfer reaction between cytb5
and apo-myoglobin:
CD spectroscopy.The transfer of heme from cyt b5to
apo-Mb was examined in the 190–250 nm and 250–500 nm
regions separately (10 mM sodium acetate buffer, pH 5.5,
room temperature) Equal volumes of cyt b5and apo-Mb
were mixed at a final concentrations of 25 lMand 30 lMfor
cyt b5 and apo-Mb, respectively The spectrum recording
conditions were the same as described above
UV–visible spectroscopy Kinetic analysis of heme
disso-ciation from the wild-type and the mutants of cyt b5were
performed as described by Hargrove et al [30] The heme
transfer reaction was monitored with a HP 8452A
diode-array spectrophotometer The temperature was controlled
at ± 0.1C with a Neslab RTE-5B circulating bath
instrument The reaction was initiated by rapidly mixing
equal volumes of solutions containing cyt b5and apo-Mb in
a tandem mixing cell with path length of 2· 0.438 cm The
final concentrations were 6 lM for cyt b5 and 25 lM for
apo-Mb in 10 mM sodium acetate buffer (pH 5.5) The
change in absorbance due to the heme transfer from cyt b5
to apo-Mb was monitored at 408 nm, which is the
maximum difference between cyt b5and metMb The heme
transfer reaction consists of two steps: the first step is the
release of the heme from cyt b5, and the second step is
the binding of apo-Mb with the heme b [30] Because the
second step is very fast (k¼ 5.8 · 105ÆM)1ÆS)1) and the
first step is the rate-determining step for the whole reaction
[31], the heme transfer reaction from cyt b5 to apo-Mb
could be treated as a first-order reaction The kinetic trace
can be described mathematically by the equation
DAt¼ DAeq(1–e–kt) where DAtis the increase in absorbance
at time t, DAeqis the increase in absorbance at equilibrium,
and k is the rate constants for heme transfer
The activation energy of the heme transfer reaction was obtained by measuring the rate constant over the temperature range of 20–37C (10 mM sodium acetate buffer pH 5.5) The activation free energy was calculated from the equation [32,33] k¼ kBT/h exp(– DG„/RT) where k is the experimental rate of heme dissociation, R is the gas constant, T is the temperature, h is the Planck constant, kBis the Boltzmann constant, and DG„is the activation free energy
R E S U L T S
Molecular structure of the cyt b5Phe35fiTyr mutant The final structure of the Phe35fiTyr mutant refined at 1.8 A˚ resolution gave an R factor of 19.2% and an Rfreeof 23.8% The root mean square (r.m.s.) deviations are 0.010 A˚ and 1.08 from the ideal bond lengths and bond angles, respectively The refinement statistics are summar-ized in Table 2 All of the nonglycine residues of the final model are located within the allowed regions (91.7% in the most favored regions) of the Ramachandran plot obtained
by running the programPROCHECK[34] The Luzzati plot shows that the estimated error of the refined coordinates is
0.21 A˚
Fig 1 shows the electron density of Tyr35 and the heme group in the Phe35fiTyr mutant The overall structure of the Phe35fiTyr mutant is basically the same as that of the wild-type Tb5 The r.m.s deviation for a total of 82 Ca atoms between the two molecules is 0.07 A˚ The secondary structures of the wild-type protein and its Phe35fiTyr mutant are the same Fig 2A and B shows a part of the heme-binding pocket of the Phe35fiTyr mutant in two different views In wild-type cyt b5, the residue Phe35 is located at helix II, which is a part of the heme-binding pocket of cyt b5, and its side-chain points toward the heme The mutation from the nonpolar residue Phe35 to the polar residue Tyr35 makes slight changes in the side chain conformation of this residue The shift of the Ca atom of Tyr35 of the Phe35fiTyr mutant from that of Phe35 of the wild-type cyt b5is 0.21 A˚, within the error limit The side chain of Tyr35 of the Phe35fiTyr mutant also points toward the heme, but the phenol ring shifts away slightly from the heme plane to avoid the unreasonable contacts with the heme The largest shift between the two superimposed Table 2 Refinement statistics.
Root-mean-square deviation
Mean temperature factors (A˚ 2 )
Trang 4aromatic rings is 0.45 A˚, i.e., the distance from the atom CZ
(Fig 1) of Tyr35 to that of Phe35 The crystal structure of
the Phe35fiTyr mutant shows that the side chain of Tyr35
makes strong van der Waals’contacts with the heme, and
the shortest distance is 3.21 A˚, i.e., from the phenol oxygen
atom of Tyr35 to the carbon atom CHB (Fig 1) of the
heme In addition, the hydroxyl group of Tyr35 forms a
hydrogen bond (2.86 A˚) to a water molecule located outside
the heme pocket, as shown in Fig 2A This water molecule
forms another hydrogen bond (2.79 A˚) with the atom ND1
of the His26 side chain in a symmetry-related molecule (Fig 2A) This water molecule was also found in the structure of wild-type cyt b5 as well as in other mutants When Phe35 is mutated to Tyr35, this water molecule moves toward the hydroxyl group of Tyr35 by 0.43 A˚, and the side chain conformation of His26 correspondingly moves a little bit (for example, the atom ND1 of His26 moves by 0.15 A˚) to be closer to the water molecule, which
is shown in Fig 2B These hydrogen-bonding interactions help to stabilize the orientation of Tyr35 side chain The
Fig 2 Stereo views of a part of the heme-binding pocket of the Phe35fiTyr mutant These diagrams were prepared using the graphics program SETOR [60] (A) Helices II, III, IV, V of Phe35fiTyr are shown as a rib-bon diagram Tyr35 and the heme group of the Phe35fiTyr mutant are shown as thick lines The water molecule (Wat) hydrogen bonded to Tyr35 is shown as a large sphere His26 of the symmetry-related molecule (#) is also shown as thick lines Hydrogen bonds are shown as broken lines Phe35 and the heme group of wild-type cyt b 5 , shown as thin lines, are superimposed with Tyr35 and the heme of the mutant (B) Tyr35 of the Phe35fiTyr mutant is superimposed with Phe35 of the wild-type cyt b 5 Heme, the water molecule and His26 # of Phe35fiTyr mutant are su-perimposed with those of the wild-type cyt b 5 Those in the mutant are shown as thick lines and large spheres, and those in wild-type cyt b 5 are shown as thin lines and small spheres (His26 # of the wild-type cyt b 5 is very close to that in the mutant and cannot be seen).
Fig 1 Stereo view of the (2Fo-Fc) electron density of Tyr35 and heme in the Phe35fiTyr mutant, contoured at 1.0 r The atoms CHB
of heme as well as OH and CZ of Tyr35 are labeled This diagram was prepared using the graphics program TURBO - FRODO
Trang 5conformation of the heme in the Phe35fiTyr mutant is
basically the same as that in wild-type cyt b5 One of the two
propionates is hydrogen bonded to the main- and side chain
atoms of Ser64, while the other one extends into the solvent
and does not form any hydrogen bond with the protein
atoms The former propionate displays the conserved
conformation in the structures of the Phe35fiTyr mutant
and of the wild-type protein as well as other mutants
However, the conformation of the latter is flexible
CD spectra of thermal denaturation of cytb5
and its Phe35fiTyr mutant
Fig 3A shows the CD spectra of wild-type cyt b5in the
far-UV region at 30C, 65 C, 70 C and 95 C, respectively
The Phe35fiTyr mutant shows similar CD spectra (data
not shown) When cyt b5and its mutant were subjected to
increasing temperature, the peak at 219 nm decreased
monotonically At 95C, the negative peak at 219 nm
almost disappeared, but a large negative peak appeared at
203 nm For wild-type cyt b5, the peak at 207 nm was still
present at 95C, but for the mutant, the peak at 208 nm
changed to a shoulder peak All of these results suggest that
the a-helix percentage of the protein and its mutant
decreases sharply while the b-sheet percentage also reduces
significantly at high temperature
Fig 3B shows the CD spectra of wild-type cyt b5in the 250–500 nm region at 30C, 67 C, 69.5 C, 75 C, 85 C, and 95C, respectively The CD spectra of the Phe35fiTyr mutant have a similar pattern and are not shown here In the Soret region, the peak positions of the two proteins are basically similar at 30C, consistent with those reported in literatures [35,36] At the near UV region, the negative CD peak at 268 nm derived from the four tyrosyl residues of wild-type cyt b5 [35] shows a different shape for the Phe35fiTyr mutant, which has five tyrosyl residues The peak at 299.4 nm derived from the single tryptophan residue for the wild-type protein shifts to 297.6 nm for the mutant The spectra in the 267–299.4 nm region are only slightly different for these two proteins
Thermal denaturation of wild-type cyt b5 and its Phe35fiTyr mutant show similar CD behavior A negative peak at 418 nm with strong intensity and a positive peak at
390 nm at room temperature are characteristic of low-spin state of ferric cyt b5 [36] When the temperature was increased the negative peak at 418 nm was blue-shifted with
a gradual reduction of its intensity Simultaneously, the intensity of the positive peak at 390 nm decreased We found that with increasing temperature to 69.5C, a new peak around 398 nm with a negative intensity appeared The intensity of the peak at 398 nm increased dramatically from 69.5 to 75C, and then gradually decreased from
75C to higher temperature However, even at 95 C, this peak does not disappear Unexpectedly our results are very different from those of the rabbit liver cyt b5reported by Sugiyama et al [36] Their work showed that there was almost no absorption in the 300–500 nm region of the CD spectrum when the temperature was 83C This is the first detailed CD spectrum study on the secondary structure
of cyt b5, and characterization of the intermediate conformation
The negative peak at 267 nm, which is attributed to absorption from tyrosyl residues Tyr6, Tyr7, Tyr27 and Tyr30 [35,37], gradually decreased with increasing tempera-ture At 69.5C, the peak intensity reduced to almost zero
A positive peak at this region appeared and its intensity gradually increased when the temperature changed from 69.5C to 75 C, then gradually decreased at higher temperature The negative peak at 299.4 nm, which is assigned to the contribution of Trp22, decreased monotoni-cally with the increase in temperature It is known from X-ray structural analysis of wild-type cyt b5 [21] that the core 2 consists of b-strand III (Tyr27–Leu32), b-strand II (Thr21–Leu25), b-strand I (Lys5–Tyr7) and a-helix I (Thr8– His15) Trp22, Tyr6, Tyr7, Tyr27 and Tyr30 are the main aromatic components of the core 2 of cyt b5 The pattern of the absorption changes around 267 nm and 299.4 nm implies that even though core 2 is largely intact after the removal of the heme from the protein as reported by Falzone et al [38] core 2 experiences significant structural fluctuation and gradually undergoes complete unfolding This study clearly shows the whole process of unfolding and
is an important supplement to the results reported by Pfeil [39] by means of second derivative spectra and heat capacity
of apo- and holo-cyt b5 Fig 4A demonstrates the transitional CD curves of wild-type cyt b5monitored at 222 nm, 299 nm, 398.4 nm and 418.8 nm The curves of 222 nm, 299 nm and 418.8 nm possess a similar pattern suggesting that dissociation of the
Fig 3 CD spectra of the wild-type cyt b 5 from 30 °C to 95 °C at (A)
195–250 nm and (B) 250–500 nm (for clarity of comparison, only part of
the spectra are shown.)
Trang 6Fe–His bond is accompanied by the a-helix unfolding of the
peptide chain and the destroying of Trp22 asymmetrical
environment Fig 4B shows the transitional curves of the
Phe35fiTyr mutant, which exhibits a pattern similar to that
of the wild-type protein All of the CD spectra transitions of
the Phe35fiTyr mutant at 222 nm, 299 nm, 398.4 nm, and
418.8 nm in response to heat are 3 C higher than those
of the wild-type cyt b5, which is consistent with the result of
UV–visible measurement [13] The results of denaturation
of these proteins by guanidine hydrochloride are also in
agreement with those of urea denaturation These results
demonstrate that the Phe35fiTyr mutant increases not only
the affinity of the heme to the polypeptide chain but also the
stability of the secondary structure
The kinetics of the heme dissociation from cytb5
variants mediated by urea
Urea-mediated denaturation of cyt b5variants was treated
as a first-order reaction, producing the rate constants of the
heme dissociation at different urea concentrations The
results are shown in Fig 5 The rate constants of heme
dissociation reaction increased slightly with the increase in
urea concentration for the wild-type protein However, it is
interesting to note that cyt b5Phe35fiTyr shows a lower
rate On the contrary, for the Phe35fiLeu mutant the rate
constant increased sharply after the urea concentration exceeded 5M These results reflect the tightness of the heme attaching to the polypeptide of cyt b5 For the Phe35fiTyr mutant the heme pocket traps the heme even more strongly than the wild-type protein Obviously, for the Phe35fiLeu mutant the interactions between the heme and its pocket are much weaker, only a moderate concentration of urea is needed to speed up the release of heme from the pocket
The heme transfer from cytb5or its Phe35fiTyr mutant
to apo-Mb The kinetic parameters of heme dissociation from cyt b5
were determined under nondenaturation conditions by measuring the spontaneous release of the heme from cyt b5
to apo-Mb, which is used as a heme trap Although the CD spectra could show the reaction process clearly, the protein concentration required is much higher than for the UV–visible method Because the high concentration of protein could cause denaturation of the apo-protein during the long assay time, all of the heme transfer reactions were monitored only by the UV–visible spectra The rates of heme transfer reaction from the wild-type or the Phe35fi Tyr mutant of cyt b5 to apo-Mb at 25 ± 0.1C show obviously differences, which can be seen in Fig 6 and Table 3 Compared with wild-type cyt b5, the heme affinity
of the Phe35fiTyr mutant increased greatly The activation free energy and activation energy listed in Table 3, which were calculated from Eyring plots (Fig 7) and Arrhenius plots (data not shown), also show that the mutation has affected the conformation of the transition state
The CD spectra of Mb, apo-Mb, wild-type cyt b5 and apo-cyt b5demonstrate that these proteins have an identical structure as reported previously [35,36,40,41] The apo-cyt b5and apo-Mb have no absorption in the Soret band region because of the lack of the heme prosthetic group For the holo-cyt b5and holo-Mb, the CD spectra of the Soret band are entirely different, which illustrates the difference in the heme environment between cyt b5and Mb In the CD spectra of cyt b5, there is a negative peak at 418 nm with strong intensity [36] In contrast, Mb has a strong positive absorption at 408 nm [41] Hence, the heme transfer reaction from cyt b to apo-Mb could be easily and precisely
Fig 4 The transitional curves of the CD spectra on heating at
222 nm, 299 nm, 398.4 nm, and 418.8 nm (A) Wild-type cyt b 5
(B) Phe35fiTyr mutant of cyt b 5
Fig 5 The rate constants of heme dissociation of cyt b 5 as function of urea concentration.
Trang 7monitored by CD which clearly demonstrates that the heme
transfer reaction under the conditions used proceeded to
completion (data not shown) Meanwhile, from the
concen-tration changes of the holo-Mb in the reaction monitored by
UV–visible spectroscopy, the same conclusion) that this
reaction is entirely completed) can be drawn
D I S C U S S I O N
Protein folding studied by CD spectra
Up to now, CD spectra of cyt b5have been studied by only
a few groups [35,36,39] These CD studies suggested that
there is an increase in disorder and less secondary structure
in the apo-form [35] However, no detailed information was
provided about the protein’s folding and stability There is evidence for the folding of apo-cyt b5in vivoprior to the formation of holo-cyt b5[42] Meanwhile, it is reported that cyt b5consists of two hydrophobic cores Core 1 is normally retained by the prosthetic heme group; core 2 comprises mainly b-sheets These two cores are well maintained in the apo-form of the protein [43] and so are especially interesting for the study of the folding mechanism, intermediates and stability of the protein by CD spectra
Usually, the stability of cyt b5 could be investigated through the heme dissociation reaction by exposing the protein to the denaturant or heat This process was considered to be a two-state mechanism (HÐ A), in which only the holo-cyt b5(H) and the apo-cyt b5(A) are present at significant concentrations [28,44] It was thought that the heme-binding domain of cyt b5 was denatured simultaneously with heme dissociation The UV–visible spectrum study of cyt b5 in response to heat and urea did display several isosbestic points in the absorbance curves, and the denaturation curves really showed that the denaturation followed the two-state mechanism
The denaturation curves of CD absorption at 222 nm,
299 nm and 418.8 nm shown in Fig 4A and B indicate that unfolding of the a-helices, b-sheets and breaking of the His–
Fe bonds of the heme follow the two-state mechanism It is noted that a new absorption peak that appeared at 398.4 nm displays slightly different denaturation behav-iours Definitely, the absorption at 398.4 nm is derived from
a heme derivative As heme is a symmetrical chromophore,
it exhibits no inherent optical activity itself [45,46] Our experiment also shows that heme in the buffer solution itself does not exhibit any CD absorption in the region of 250–
500 nm at 30–95C Apo-cyt b5has no CD absorption in the Soret band too, but shows the absorption contributed from aromatic amino acids in the near UV region [35] The concurrent existence of the Soret band absorption at 418.8 nm and 398.4 nm at 69.5C shown in Fig 3B indicates that probably there are two types of heme derivative in the solution; at this stage the heme was not totally released from the protein heme pocket into the aqueous environment and part of the low-spin and six-coordinated heme was changed into the high-spin state
Fig 7 Eyring plots of the rate constants of heme transfer from cyt b 5 to apo-myoglobin; (j) Phe35fiTyr mutant (d) wild-type cyt b 5
Table 3 The kinetic parameters of the heme-transfer reactions between
apo-Mb and the wild-type and the Phe35fiTyr mutant of cyt b 5 The
measurements were made in sodium acetate buffer, I ¼ 10 m M ,
pH 5.5.
a T ¼ 25 ± 0.1 C.
Fig 6 Kinetic traces for heme transfer reaction from the wild-type, or
Phe35fiTyr mutant of cyt b 5 to apo-myoglobin (A) Experimental data.
(B) Fitted curve.
Trang 8with breaking of the His–Fe bonds It is known that the
apo-cyt b5prepared under mild conditions could generally
maintain the holo-like structures except for some
confor-mational fluctuations observed in the local regions [47]
However, as indicated by molecular dynamics simulations
all a-helices in core 1 are highly mobile, and the tertiary
structure in core 2 of cyt b5 is rather rigid [48] Thus, the
denaturation curve of the wild-type protein monitored at
398.4 nm and 67–75C by CD implied that there was
probably a collapse of core 1 accompanied by partially
unfolding of the a-helices and breaking of Fe–His bonds
This temperature region is coincident with the transition
region of cyt b5denaturation in response to heat monitored
by UV–visible spectra at 418 nm At this time, the heme was
still wrapped up in the polypeptide chain of cyt b5
Therefore, the CD absorption at 398.4 nm could be the
result of another form of heme, an intermediate state, in
which the heme is not coordinated by two histidine residues
and does not sit normally in the heme pocket More
probably it is enveloped by the partially unfolded cyt b5
polypeptide chain after the collapse of hydrophobic core 1
From the observations of CD absorption of tryptophan and
tyrosines, however, it is believed at that time the core 2 of
cyt b5remains intact Even at 95C, this peak at 398.4 nm
does not disappear completely Possibly, the heme is still
partially attached to some parts of the random coil of
denatured cyt b5 polypeptide chain through hydrophobic
interactions Actually, after we reached this conclusion, we
found that Gray’s group had published a short
communi-cation indicating that in the folding study of cyt b562,
normally the heme iron is ligated axially by the side chains
of Met7 and His102 It is likely that one of these ligands
remains attached to the heme in the unfolded state [49,50]
Here, we provide the detailed CD spectra evidencing the
existence of the intermediate and a reasonable explanation
The reason why our results do not agree with those
obtained for the rabbit liver cyt b5[36] is not yet known
But, it is noted that the bovine liver Tb5used in this work is
more stable than rabbit liver cyt b5 The Tm (transition
midpoint of the heat denaturation curve of the UV–visible
spectrum at 412 nm) is 66.9C for bovine liver Tb5 and
55.0C for rabbit liver cyt b5 [13,36] Maybe a detailed
structural study, similar to the comparison between the
microsomal cyt b5and the outer membrane liver
mitochon-dria cyt b5 [51], is required to reveal the essence of the
different properties
The stability of cytb5Phe35 mutants
The wild-type protein usually develops an optimal
archi-tecture to fulfill its biological functions after hundreds and
thousands years of evolution and natural selection
Arti-ficial site-directed mutagenesis of proteins most often leads
to a decrease in stability: an increase in stability in the
mutant proteins is comparatively rare [52,53] The main
components of protein stability that could be perturbed by
mutation at interior groups include hydrophobic effects,
van der Waals’forces, backbone conformation, hydrogen
bonds, local polarity and side chain volume of the
substituted residue Substitution of tyrosine for
phenyl-alanine should generate 4.8 kJÆmol)1destabilization energy
because of the decreased hydrophobic nature of tyrosine,
and may contribute 4–6 kJÆmol)1 to protein stability if
there is another hydrogen bond generated in the cyt b5 Phe35fiTyr mutant [53,54] In our previous study [13], the Phe35fiTyr mutant of cyt b5 in the oxidized state is 3.3 kJÆmol)1 more stable than the wild-type protein towards heat denaturation and is 4.3 kJÆmol)1more stable
in urea denaturation The CD spectra of heat denaturation also show that the structure transition temperature for the Phe35fiTyr mutant is higher than that for the wild-type Kinetically, the rate constant of heme transfer reactions from cyt b5to apo-Mb for the wild-type protein is 10 times faster than that for the Phe35fiTyr mutant The urea-mediated heme dissociation reactions of various cyt b5
variants also demonstrate that the heme is trapped in the heme pocket with different degrees of tightness Recently, Silchenko et al [55] found that cyt b5 from the outer mitochondrial membrane of rat liver is substantially more stable against thermal and chemical denaturation than bovine liver cyt b5 Their study demonstrated that the enhanced stability of outer mitochondrial membrane cyt b5
is in large part due to slow heme release, where the heme is kinetically trapped in the heme pocket of hemoproteins As shown in previous work, the residues on the protein surface were considered to be less important and to have minor effect on protein stability because these residues exert little effect on the interactions between the heme and the heme pocket [6,56] For the heme pocket residues such as Phe35, Val61, Val45 and Phe58 the situation is entirely different The mutation from the hydrophobic residue Phe35 to a larger polar residue Tyr35 does not make significant changes in the overall structure and the local structure around the mutation site because there is enough space to accommodate an additional hydroxyl group However, the crystal structure of the Phe35fiTyr mutant shows that the hydroxyl oxygen atom of the side chain of Tyr35 is 3.21 A˚ away from the atom CHB of the pyrrole group of the heme, making strong van der Waals’contacts with the heme Obviously, the introduction of hydroxyl group
in the heme pocket strengthens the interactions between Tyr35 and the heme with the iron in the oxidative state The increased van der Waals’interactions between the side chain of Tyr35 and the heme can probably make an obstacle to the departure of the heme from the hydropho-bic pocket of the protein The total consequence of this mutation made ferricytochrome b5 Phe35fiTyr more stable compared with the wild-type protein In the case
of the cyt b5 Phe35fiHis mutant, besides the increased hydrophilicity of the histidine residue, the side chain volume decreases by 36 A˚3 compared to the wild-type cyt b5 which would effectively reduce the van der Waals’ contact between the histidine and the heme So, the Phe35fiHis mutant is 11.8 kJÆmol)1 less stable than the wild-type protein [13]
There is a stabilization effect of the heme ring binding to Phe35 and Phe58 by hydrophobic aromatic interactions It has been reported that an edge-to-face orientation between two aromatic groups is energetically favorable [57] Sakamoto et al [45] have studied the effect of amino acids substitution of hydrophobic residues on heme-binding properties in the designed two-a-helix peptides Their studies demonstrated that the edge-to-face interactions between the aromatic side chain of the phenylalanine residues and the porphyrin plane might contribute to the conformation of peptide–heme conjugates They also
Trang 9proved that the phenylalanine residue located at i ± 4
relative to the axial ligand histidine residue in the a-helix was
critical to the edge-to-face interaction between the
phenyl-alanine side chain and the porphyrin ring, providing
stabilization of peptide–heme conjugates [45,46,58] The
Phe35–His39 of cyt b5is consistent with i ± 4 arrangement
It is clear that the substitution of tyrosine for phenylalanine
at position 35 does not destroy the aromatic interactions
and can also maintain the edge-to-face interaction,
provi-ding the stabilization effect of the heme binprovi-ding In the case
of the Phe35fiLeu mutant, however, substitution of leucine
for phenylalanine should break this effect This is also
supported by the denaturation experiment [13], which
showed that the Phe35fiLeu mutant is 7.8 kJÆmol)1less
stable towards heat and 7.9 kJÆmol)1less stable towards
urea than the wild-type protein
Factors affecting redox potential of the Phe35 mutants
The redox potential of the Phe35fiTyr mutant shifts
negatively by 66 mV compared to that of the wild-type
cyt b5 [13] As we know, a hydrophilic environment
stabilizes the oxidized state, leading to a lower redox
potential [53] In particular, the introduction of a polar
hydroxyl group in the low dielectric interior of the protein
can play a much stronger electrostatic role, stabilizing ferric
iron The reduction of hydrophobicity reasonably accounts
for the negative shift of redox potential In addition, the
hydrogen bonding formation between the tyrosine and the
conserved water molecule shown in the crystal structure of
the Phe35fiTyr mutant enhances significantly hydrophilic
influence on the heme causing great alteration of the protein
properties [59]
A C K N O W L E D G M E N T S
This work was supported by two grants from the National Natural
Science Foundation of China We are grateful to Prof Li-Wen Niu,
Prof Mai-Kun Teng and Dr Xue-Yong Zhu of the University of
Science and Technology of China for their support and help with the
X-ray data collection.
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