X-ray crystallography, CD and kinetic studies revealed the essence of the abnormal behaviors of the cytochrome b 5 Phe35fiTyr mutant Ping Yao 1 , Jian Wu 2 , Yun-Hua Wang 1 , Bing-Yun Sun 1 , Zong-Xiang Xia 2 and Zhong-Xian Huang 1 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 b 5 (cyt b 5 ). This patch is partially exposed on the surface of cyt b 5 while 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 b 5 :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 b 5 and the oxidized Phe35fiTyr mutant is more stable towards thermal and chemical denaturation than wild-type cyt b 5 . 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 b 5 ; folding; mutagenesis; stability; structure. Cytochrome b 5 (cyt b 5 ) 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 b 5 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 b 5 , 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 b 5 , 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 b 5 , 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 b 5 ,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 b 5 [14], and the oxidized Phe35fiTyr mutant is obviously more stable towards heat and chemical denatur- ationthanwild-typecytb 5 [13]. We also studied electron transfer reactions of cyt b 5 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(NH 3 ) 6 ] 3+ . The change at Phe35 of cyt b 5 did not affect the second-order rate constants of the electron transfer Correspondence to Z X.Huang,ChemicalBiologyLaboratory, 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:cytb 5 :cytochromeb 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.YaoandJ.Wumadeequalcontributionstothiswork. 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) Eur. J. Biochem. 269, 4287–4296 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03120.x reactions. 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 theessentialdifferencebetweenthewild-typeandmutant proteins and to give a proper interpretation. In this paper, the secondary structural changes of cyt b 5 and 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 b 5 or its Phe35fiTyr mutant and apo-myoglobin (Mb) were studied to demonstrate the affinity changes of the heme with cyt b 5 polypeptide chain. In this paper the crystal structure of the cyt b 5 Phe35fiTyr 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. MATERIALS AND METHODS Protein preparation Bovine liver cyt b 5 and its mutants were prepared and purified as described previously [13]. The concentrations of ferricytochrome b 5 and the mutants were determined with the value of OD 414 ¼ 117 m M )1 Æcm )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 e 409 ¼ 171 m M )1 Æcm )1 [19], and e 280 ¼ 15.2 m M )1 Æcm )1 [20], respectively. X-ray analysis of cytochrome b 5 Phe35fiTyr mutant Crystallization. Single crystals of the Phe35fiTyr mutant of trypsin-solubilized bovine liver microsomal cytochrome b 5 (Tb 5 ) were grown by the vapor diffusion method in hanging drops containing 10 mgÆmL )1 protein solution in 3.1–3.2 M phosphate buffer (pH 7.5) at 20 °C. This is similar to the crystallizing condition used for wild-type Tb 5 [21] and lipase-solubilized bovine liver microsomal cyto- chrome b 5 (Lb 5 ) [22]. The typical size of the single crystals was  0.6 · 0.5 · 0.3 mm. Crystals of wild-type Tb 5 [21] and the Tb 5 Val61fiHis mutant [23] are isomorphous belonging to the monoclinic space group C2 with the following unit cell parameters: a ¼ 70.71 A ˚ ,b¼ 40.39 A ˚ , c ¼ 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 programs DENZO and SCALEPACK [24], giving an R sym of 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 5 Phe35fiTyr mutant were carried out using the program packages X - PLOR [25] and CNS [26] successively on a Silicon Graphics Indigo 2 workstation. All the data up to 1.8 A ˚ were used for structural refinement at the CNS refinement 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 b 5 at 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 R free of 28.4%. The positional refinement and temperature factor refinement were carried out for each round using the program X - PLOR . The program TURBO - FRODO was 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 peakshigherthan3 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 package CNS . 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 cyt b 5 monitored by CD CD spectra of cyt b 5 and 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. Space group C2 Cell dimensions a(A ˚ ) 70.71 b(A ˚ ) 40.39 c(A ˚ ) 39.30 b (°) 111.72 Number of molecules per asymmetric unit 1 Vm (A ˚ 3 ÆDa )1 ) 2.46 Resolution (A ˚ ) 1.8 Number of unique reflections 9129 R sym (%) a 6.1 (31.5) b Data completeness (%) 94.3 (79.7) b 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 ˚ ). c Mean signal-to-noise ratio. 4288 P. Yao et al. (Eur. J. Biochem. 269) Ó FEBS 2002 respectively. The ellipticity was recorded at 100 nmÆmin )1 speed, 0.2 nm resolution, five accumulations, 1.0 nm bandwidth. Cyt b 5 or its mutant was dissolved in the phosphate buffer (100 m M pH 7.0). The protein concentra- tions were 25 l M in the 190–250 nm region and 12.5 l M in 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 30–95 °Candthe temperature accuracy was within ± 0.1 °C. Urea- and guanidine hydrochloride-mediated denaturation of cyt b 5 variants For the kinetic study of urea-mediated denaturation of cyt b 5 and its variants, the time course of the absorbance increase at 412 nm was recorded immediately after mixing of 0.3 mL cyt b 5 and 2.7 mL urea or guanidine hydrochlo- ride at 30 °C. The protein solution was prepared in a 100 m M phosphate buffer (pH 7.0). The final concentration of cyt b 5 was 4 l M , and the concentration of urea and guanidine hydrochloride varied from 0 to 10 and from 0 to 6 M , respectively. All measurements were carried out on a HP 8452A diode-array spectrophotometer (Hewlett-Pac- kard). The kinetics of heme dissociation from cyt b 5 by urea was analyzed as described in the literature [28,29]. Heme-transfer reaction between cyt b 5 and apo-myoglobin: CD spectroscopy. Thetransferofhemefromcytb 5 to apo- Mb was examined in the 190–250 nm and 250–500 nm regions separately (10 m M sodium acetate buffer, pH 5.5, room temperature). Equal volumes of cyt b 5 and apo-Mb were mixed at a final concentrations of 25 l M and 30 l M for cyt b 5 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 b 5 were performed as described by Hargrove et al.[30].Theheme transfer reaction was monitored with a HP 8452A diode- array spectrophotometer. The temperature was controlled at ± 0.1 °C with a Neslab RTE-5B circulating bath instrument. The reaction was initiated by rapidly mixing equal volumes of solutions containing cyt b 5 and apo-Mb in a tandem mixing cell with path length of 2 · 0.438 cm. The final concentrations were 6 l M for cyt b 5 and 25 l M for apo-Mb in 10 m M sodium acetate buffer (pH 5.5). The change in absorbance due to the heme transfer from cyt b 5 to apo-Mb was monitored at 408 nm, which is the maximum difference between cyt b 5 and metMb. The heme transfer reaction consists of two steps: the first step is the release of the heme from cyt b 5 , and the second step is the binding of apo-Mb with the heme b [30]. Because the second step is very fast (k ¼ 5.8 · 10 5 ÆM )1 ÆS )1 )andthe first step is the rate-determining step for the whole reaction [31], the heme transfer reaction from cyt b 5 to apo-Mb could be treated as a first-order reaction. The kinetic trace can be described mathematically by the equation DA t ¼ DA eq (1–e –kt )whereDA t is the increase in absorbance at time t, DA eq is 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 20–37 °C(10m M sodium acetate buffer pH 5.5). The activation free energy was calculated from the equation [32,33] k ¼ k B T/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, k B is the Boltzmann constant, and DG° „ is the activation free energy. RESULTS Molecular structure of the cyt b 5 Phe35fiTyr mutant The final structure of the Phe35fiTyr mutant refined at 1.8 A ˚ resolution gave an R factor of 19.2% and an R free of 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 program PROCHECK [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 Tb 5 . 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 b 5 , the residue Phe35 is located at helix II, which is a part of the heme-binding pocket of cyt b 5 , 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 b 5 is 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. No. of amino acid residues 82 No. of prosthetic group 1 No. of solvent molecules 94 No. of reflections used 8870 R factor (%) 19.2 Free R factor (%) 23.8 Root-mean-square deviation Bond lengths (A ˚ ) 0.010 Bond angles (°) 1.08 Mean temperature factors (A ˚ 2 ) Main chain 22.11 Side chain 26.29 Heme 23.78 Solvent 42.16 Ó FEBS 2002 Mutation at Phe35 of cytochrome b 5 (Eur. J. Biochem. 269) 4289 aromatic 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 b 5 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 Phe35fiTyrmutantareshownasthick 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 ,shownasthinlines, 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 . 4290 P. Yao et al. (Eur. J. Biochem. 269) Ó FEBS 2002 conformation of the heme in the Phe35fiTyr mutant is basically the same as that in wild-type cyt b 5 . 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 cyt b 5 and its Phe35fiTyr mutant Fig. 3A shows the CD spectra of wild-type cyt b 5 in the far- UV region at 30 °C, 65 °C, 70 °Cand95°C, respectively. The Phe35fiTyrmutantshowssimilarCDspectra(data not shown). When cyt b 5 and its mutant were subjected to increasing temperature, the peak at 219 nm decreased monotonically. At 95 °C, the negative peak at 219 nm almost disappeared, but a large negative peak appeared at 203 nm. For wild-type cyt b 5 , the peak at 207 nm was still present at 95 °C, 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 b 5 in the 250–500 nm region at 30 °C, 67 °C, 69.5 °C, 75 °C, 85 °C, and 95 °C, 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 30 °C, 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 b 5 [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 b 5 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 b 5 [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 69.5 °C, 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 75 °C, and then gradually decreased from 75 °C 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 b 5 reported 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 83 °C. This is the first detailed CD spectrum study on the secondary structure of cyt b 5 , 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 69.5 °C, the peak intensity reduced to almost zero. A positive peak at this region appeared and its intensity gradually increased when the temperature changed from 69.5 °Cto75°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 b 5 [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 b 5 . 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 b 5 . Fig. 4A demonstrates the transitional CD curves of wild-type cyt b 5 monitored 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 °Cto95°Cat(A) 195–250 nm and (B) 250–500 nm (for clarity of comparison, only part of the spectra are shown.) Ó FEBS 2002 Mutation at Phe35 of cytochrome b 5 (Eur. J. Biochem. 269) 4291 Fe–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 ofthewild-typecytb 5 , 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 cyt b 5 variants mediated by urea Urea-mediated denaturation of cyt b 5 variants 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 b 5 Phe35fiTyrshowsalower rate. On the contrary, for the Phe35fiLeu mutant the rate constant increased sharply after the urea concentration exceeded 5 M . These results reflect the tightness of the heme attaching to the polypeptide of cyt b 5 . 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 cyt b 5 or its Phe35fiTyr mutant to apo-Mb The kinetic parameters of heme dissociation from cyt b 5 were determined under nondenaturation conditions by measuring the spontaneous release of the heme from cyt b 5 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 Tyrmutantofcytb 5 to apo-Mb at 25 ± 0.1 °C show obviously differences, which can be seen in Fig. 6 and Table 3. Compared with wild-type cyt b 5 , 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 b 5 and apo-cyt b 5 demonstrate that these proteins have an identical structure as reported previously [35,36,40,41]. The apo- cyt b 5 and apo-Mb have no absorption in the Soret band region because of the lack of the heme prosthetic group. For the holo-cyt b 5 and holo-Mb, the CD spectra of the Soret band are entirely different, which illustrates the difference in the heme environment between cyt b 5 and Mb. In the CD spectra of cyt b 5 , 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 5 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. 4292 P. Yao et al. (Eur. J. Biochem. 269) Ó FEBS 2002 monitored 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. DISCUSSION Protein folding studied by CD spectra Up to now, CD spectra of cyt b 5 have 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 b 5 in vivo prior to the formation of holo-cyt b 5 [42]. Meanwhile, it is reported that cyt b 5 consists 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 b 5 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 (H Ð A), in which only the holo-cyt b 5 (H) and the apo-cyt b 5 (A) are present at significant concentrations [28,44]. It was thought that the heme-binding domain of cyt b 5 was denatured simultaneously with heme dissociation. The UV–visible spectrum study of cyt b 5 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 30–95 °C. Apo-cyt b 5 has 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 69.5 °CshowninFig.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. Wild-type Phe35fiTyr k (h )1 ) a 2.01 (± 0.02) 0.21 (± 0.03) DG° „ (kJÆmol )1 ) 91.2 96.8 E a (kJÆmol )1 ) 110.4 135.4 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. Ó FEBS 2002 Mutation at Phe35 of cytochrome b 5 (Eur. J. Biochem. 269) 4293 with breaking of the His–Fe bonds. It is known that the apo-cyt b 5 prepared 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 b 5 is rather rigid [48]. Thus, the denaturation curve of the wild-type protein monitored at 398.4 nm and 67–75 °C 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 b 5 denaturation 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 b 5 . 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 b 5 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 b 5 remains intact. Even at 95 °C, 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 b 5 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 b 562 , normally Ôthe 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 stateÕ [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 b 5 [36] is not yet known. But, it is noted that the bovine liver Tb 5 used in this work is more stable than rabbit liver cyt b 5 .TheT m (transition midpoint of the heat denaturation curve of the UV–visible spectrum at 412 nm) is 66.9 °C for bovine liver Tb 5 and 55.0 °C for rabbit liver cyt b 5 [13,36]. Maybe a detailed structural study, similar to the comparison between the microsomal cyt b 5 and the outer membrane liver mitochon- dria cyt b 5 [51], is required to reveal the essence of the different properties. The stability of cyt b 5 Phe35 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 )1 destabilization 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 b 5 Phe35fiTyr mutant [53,54]. In our previous study [13], the Phe35fiTyr mutant of cyt b 5 intheoxidizedstateis 3.3 kJÆmol )1 more stable than the wild-type protein towards heat denaturation and is 4.3 kJÆmol )1 more 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 b 5 to 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 b 5 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 b 5 from the outer mitochondrial membrane of rat liver is substantially more stable against thermal and chemical denaturation than bovine liver cyt b 5 . Their study demonstrated that the enhanced stability of outer mitochondrial membrane cyt b 5 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 b 5 Phe35fiTyr more stable compared with the wild-type protein. In the case of the cyt b 5 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 b 5 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 4294 P. Yao et al. (Eur. J. Biochem. 269) Ó FEBS 2002 proved 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 b 5 is 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 binding. 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 )1 less stable towards heat and 7.9 kJÆmol )1 less 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 b 5 [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 Ôlow dielectricÕ 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]. ACKNOWLEDGMENTS This work was supported by two grants from the National Natural Science Foundation of China. 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X-ray crystallography, CD and kinetic studies revealed the essence of the abnormal behaviors of the cytochrome b 5 Phe35fiTyr mutant Ping Yao 1 ,. 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