SUPPLEMENTARY INFORMATION

Transcription

1 This supplementary information is an extension of the letter with the same title and includes further discussion on the comparison of our designed Fe B Mb (computer model and crystal structure) with the crystal structure of the closely related enzyme cytochrome c oxidase (Fig. S1), EPR characterization (Fig. S2), electrochemical redox potential determination of the heme reduction potential (Fig. S3), UV-vis characterization (Fig. S4), crystallographic parameters (Fig. S5, Table S1), NO activity studies by GC/MS (Fig. S6- S8) and references. Comparing designed Fe B Mb to the native protein To test how closely our designed protein mimics native NOR, the minimized Fe(II)- Fe B Mb model was overlaid with the crystal structure of a closely related enzyme, cytochrome c oxidase (CcO), as there is currently no available three-dimensional structure for bacterial NOR (Fig. S1A). From this overlay, it can be seen that the heme and proximal histidines overlay well with one another. The Zn(II) ion (as an Fe(II) mimic) in the Fe(II)-Fe B Mb model is within close vicinity of the Cu ion (1.84 Å apart) of the Cu B site of CcO. The introduced E68 is pointing towards the heme pocket and Zn(II) ion, suggesting that it can act as a ligand to the designed Fe B site. Although the three distal His from the designed protein do not overlay as well with those of CcO, they all point toward the Zn(II) ion, indicating that in combination with E68, all four ligands can help bind and support the introduction of a designed Fe B site in myoglobin. The crystal structure of Fe(II)-Fe B Mb was also overlaid with the crystal structure of CcO, as shown in Fig. S1B. Again, the hemes, proximal histidines, and two of the three distal histidines all overlay well with one another. In addition, the Fe(II) of Fe(II)-Fe B Mb and the Cu B site of CcO are located in vicinity of one another (2.55 Å). The similarities between the crystal structures of Fe(II)-Fe B Mb and CcO help provide strong evidence for the successful rational design of a metalloprotein. 1

2 A) H64 H29 Zn Cu E68 H43 H93 B) H64 Fe Cu H29 E68 H43 H93 Fig. S1. A) Overlay of the active sites of a minimized computer model of designed Fe(II)- Fe B Mb (yellow; Fe(II) modeled as Zn(II), grey sphere) with the crystal structure of bovine 2

3 heart CcO (purple; Cu(II) is shown as a brown sphere). B) Overlay of the crystal structure of Fe(II)-Fe B Mb (1.72 Å resolution; cyan; Fe(II) is shown as a green sphere) with that of the crystal structure of bovine CcO (purple). The Cu Fe distance of CcO is 4.85 Å (PDB entry 2OCC) 42. The residue numbers refer to myoglobin positions (i.e., not CcO numbering). EPR characterization of iron bound Fe B Mb After binding Fe 2+, Fe(II)-Fe B Mb was oxidized in the presence of Azurin. Fig. S2A shows spin-coupling between the heme iron(iii) and the non-heme iron(iii) of our designed Fe B Mb as evidenced by the decrease of the high spin Fe signals centered at g~6. Control experiments of wtmb (Fig. S2B) as well as Fe B Mb(-Glu) (Fig. S2C) show little change in the high spin heme signal upon iron addition and oxidation, providing further evidence that this spin-coupling is unique to Fe B Mb where Glu68 plays a crucial role in iron binding. Since Cu + is EPR silent, the decrease of Azurin Cu 2+ signals at g~2.05 (Fig. S2B and 2C) also suggest that Azurin can efficiently oxidize Fe 2+ to Fe 3+, while itself being reduced. In addition, the EPR signal of free to Fe 3+ (g = 4.26) was also observed in Fig. S2D. 3

4 A) a) g=6.69 Fe B Mb + 0 eq Fe eq Azurin b) c) g=5.01 Fe B Mb + 0 eq Fe eq Azurin Fe B Mb eq Fe eq Azurin d) Fe B Mb eq Fe eq Azurin e) Fe B Mb eq Fe eq Azurin f) Fe B Mb eq Fe eq Azurin Magnetic field (Gauss) B) 5.86 wtmb + 0 eq Fe eq Azurin wtmb + 2 eq Fe eq Azurin Magnetic field (Gauss) 4

5 C) 6.06 Fe B Mb(-Glu) + 0 eq Fe eq Azurin Fe B Mb(-Glu) + 2 eq Fe eq Azurin Magnetic field (Gauss) D) 2 eq Fe eq Azurin Magnetic field (Gauss) Fig. S2. A) EPR spectra of a) deoxy Fe B Mb (0.5 mm) only and in the presence of different eq of Fe 2+, b) 0 eq, c) 0.5 eq, d) 1.5 eq, e) 1.5 eq and f) 2.0 eq after oxidization 5

6 by 3 eq Azurin. Control experiments for deoxy wtmb B), Fe B Mb(-Glu) C) or Fe 2+ only D) were performed under identical conditions. Spectra were collected in 50 mm Bis Tris ph 7.0 at 4 K, 2 mw, and 9.05 GHz microware frequency. Electrochemical redox potential determination (by spectroelectrochemistry) Representative spectroelectrochemical titrations of deoxy Fe B Mb in the absence or presence of Fe 2+ are shown in Fig. S3. In addition to EPR evidence (Fig S2), the significant increase (>100 mv) in heme reduction potential in the presence of Fe 2+ also support the occurrence of spin-coupling between the heme iron and non-heme Fe B sites of designed Fe B Mb. 6

7 A) 1.2 Absorbance (AU) Wavelength (nm) 0.8 B) 0.6 Absorbance (AU) Wavelength (nm) Fig. S3. Spectroelectrochemical titration of A) deoxy Fe B Mb (-158 ± 4 mv vs. NHE) and B) deoxy Fe B Mb + Fe 2+ (-46 ± 2 mv vs. NHE) at ph 7. 7

8 UV-vis of oxidized Fe B Mb To identify the end product of the reaction of Fe B Mb and NO, excess NO was incubated with the oxidized met (i.e., Fe(III) heme) form of Fe B Mb. When excess NO gas was added to degassed met Fe B Mb in a sealed cuvette, the Soret band undergoes a red shift from 406 nm to 410 nm and the visible region absorbance at 619 nm decreases significantly with concomitant appearance of visible bands at 540 nm and 579 nm, which are characteristic of the formation of a six coordinate NO bound heme species 43 (Fig. S4, black spectrum). The Soret band is a good indicator of the oxidation state and ligand binding properties of the heme iron. A shorter wavelength Soret band absorbance (~400 nm) is indicative of an oxidized Fe(III) heme and a longer wavelength Soret band (~430 nm) is indicative of a five coordinate reduced Fe(II) heme. An intermediate Soret absorbance (~ nm) is characteristic of a six coordinate reduced heme species 43. The Soret band of met Fe B Mb with excess NO is located at 410 nm, which is more typical of a six coordinate oxidized low spin heme species. In addition, the visible region provides further evidence of a typical six coordinate heme nitrosyl species (i.e., similar visible region peaks to Fe(II) heme nitrosyl as shown in Fig. 2Ba, black spectrum), suggesting that Fe B Mb can bind NO in the oxidized state. Therefore, the product of NO addition to Fe(II)-Fe B Mb results in a single species that is likely the met-no form. 8

9 Met Fe B Mb + NO Met Fe B Mb Fig. S4. UV-vis spectra of met Fe B Mb and NO bound met Fe B Mb after 6000 s (1.67 hr) of incubation with NO in 50 mm Bis Tris ph 7. X-ray crystallographic studies The electron density (2F o -F c ) map of the active site in Fe(II)-Fe B Mb is shown in Fig. S5, which demonstrates the binding of Fe 2+ in the Fe B site as designed. The crystal and refinement data are summarized in Table S1. The resulting crystal structure was checked by Procheck 44, with all the residues in allowed regions. An overlay of the crystal structures of Fe(II)-Fe B Mb with native CcO is shown in Fig. S1B. 9

10 Fig. S5. Electron density (2F o -F c ) map of Fe B binding site in the distal heme pocket of Fe(II)-Fe B Mb. The final 2F o -F c Fourier map is contoured at 5.0σ level for the Fe B II area. 10

11 Table S1. Crystal and refinement data for Fe(II)-Fe B Mb. Data collection Wavelength of data collection (Å) (Fe-edge adsorption) Space Group Cell dimension a, b, c (Å) Fe(II)-Fe BMb P , 48.2, , 90.0, 90.0 α, β, γ (o) Resolution (Å) 1.72 ( ) R-merge (0.397) I/σI 27.9(4.8) Redundancy 10.9 (8.8) Completeness (%) 98.3(95.3) Refinement Resolution (Å) Rwork/Rfree 0.243/0.274 No. reflections 13,961 No. atoms Protein 1,219 Hem/Ion 42/2 FE Water 136 B-factor Protein 28.0 Hem/Ion 24.9/33.4 Water 38.4 Rms. Deviations Bond lengths(å) Bond angles ( ) NO activity studies by GC/MS The product of the reaction of Fe(II)-Fe B Mb with NO was investigation by GC/MS (Fig. 3). Typical ESI (electrospray ionization) mass spectra of NO (MW 30) and N 2 O (MW 44) are 11

12 presented in Fig. S6 and are comparable to those obtained from the NIST (National Institute of Standards and Technology) database. Fig. S6. Typical mass spectra of NO (MW 30) and N 2 O (MW 44). 12

13 Figure S7 shows the total ion chromatograms (Fig. S7, black traces) and the single ion chromatograms of only the 44 MW component (Fig. S7, blue traces), representing the main ion peak of N 2 O for the reaction of Fe(II)-Fe B Mb and of Fe 2+ only with NO. From a comparison of their intensities, the extent of N 2 O formation by Fe(II)-Fe B Mb is clearly shown in the single ion chromatogram where Fe(II)-Fe B Mb produced ~ 2 times more N 2 O than the control sample without protein. A) B) Fe(II)-FeBMb Fe 2+ only 20 hr 2 eq dithionite 20 hr 2 eq dithionite 44 MW N 2 O 44 MW N 2 O Fig. S7. A comparison of the total ion chromatogram (black traces) and single ion chromatogram of the 44 MW component (main peak of N 2 O; blue traces) at 20 hr (2 hr after dithionite addition) of the reaction of Fe(II)-Fe B Mb A) or Fe 2+ only B) with NO. 13

14 Comparison of UV-vis and GC/MS experiments of NO activity The two experiments producing Fig. 2 and Fig. 3 are different and thus on a different time frame. The experiment in Fig. 2 is a single turnover UV-vis experiment that measures the reduction of the substrate NO in solution, while the GC/MS experiment in Fig. 3 measures product (N 2 O) formation in the head space of the reaction container. For this reaction, the reduction of NO occurs first, followed by N 2 O formation in solution and then release of N 2 O into the head space for GC/MS detection. Since N 2 O has a high solubility in water (0.112 g in 100 g water or 25.5 mm at room temperature), the product N 2 O is dissolved in the solution first until it reaches its saturation point in the solution before it can be released into the head space for GC/MS detection. For this reason, it will take much longer for GC/MS to detect N 2 O in the head space than for UV-vis (to detect activity) in solution. To demonstrate this important difference, we have carried out the following experiment: we prepared a 0.18 mm N 2 O solution (equal to the maximum yield of N 2 O generated from the designed Fe B Mb in the GC/MS experiment described in Fig. 3 after two turnovers) from a saturated N 2 O solution (prepared by adding N 2 O gas into a buffer solution and letting it equilibrate for 2 hr). Upon transfer of the N 2 O saturated solution (~25 mm N 2 O) into another buffer container that does not contain any N 2 O gas but contains a similar amount of NO gas in the head space to mimic the GC/MS experimental conditions in Fig. 3 and after accounting for proper dilution to reach 0.18 mm of N 2 O in solution, we started monitoring by GC/MS the head space of this container. As seen in Fig. S8, little N 2 O gas was detected by GC/MS from the head space of the container after 1 hr of incubation, despite the fact that we had the N 2 O in the solution to begin with. After 14 hr, we were able to detect similar amounts of N 2 O in the head space as in our GC/MS experiments in Fig. 3. To allow faster detection of N 2 O in the head space, we had to use 25 mm protein (for one turnover) to reach a saturation 14

15 concentration of 25 mm N 2 O; however, it is difficult to prepare proteins with concentrations higher than 2.5 mm (the myoglobin mutant tends to precipitate at such a high concentrations). This experiment showed that GC/MS should be used only for product identification and extent of reaction, not for rate measurement under 1-2 turnover conditions. A) 1 hr B) 14 hr N 2 O N 2 O Fig. S8 GC/MS measurement of the head space gas of 50 mm Bis Tris ph 7 after adding a solution of 0.18 mm N 2 O for 1 hr A) and 14 hr B). 15

16 References 42. Yoshikawa, S. et al. Redox-coupled crystal structural changes in bovine heart cytochrome c oxidase. Science 280, (1998). 43. Antonini, E. & Brunori, M. Hemoglobin and Myoglobin in their Reactions with Ligands, 445 (Elsevier, New York, N. Y, 1971). 44. Laskowski, R. A., MacArthur, M. W., Moss, D. S. & Thornton, J. M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, (1993). 16