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Fig. S1: SDS PHAGE patterns of purified wild-type and mutant proteins demonstrate more than 90% homogeneous for proteins used in this work. 20KD Ec DOS-PAS.

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Presentation on theme: "Fig. S1: SDS PHAGE patterns of purified wild-type and mutant proteins demonstrate more than 90% homogeneous for proteins used in this work. 20KD Ec DOS-PAS."— Presentation transcript:

1 Fig. S1: SDS PHAGE patterns of purified wild-type and mutant proteins demonstrate more than 90% homogeneous for proteins used in this work. 20KD Ec DOS-PAS 12KD WT M95A M95H M95L R97I R97A R97E Marker

2 x Wavelength (nm) Absorbance A Wavelength (nm) Absorbance B Wavelength (nm) Absorbance C Fig. S Wavelength (nm) Absorbance D x (418) (417)

3 x Wavelength (nm) Absorbance E Wavelength (nm) Absorbance F G Wavelength (nm) Absorbance x5 Fig. S2. Selected spectra of 1  M wild-type Ec DOS-PAS (A), M95A (B), M95H (C), M95L (D), R97A (E), R97E (F) and R97I (G) proteins formed by adding 200  M Na 2 S under aerobic conditions. Black, blue and red lines represent the His-Fe(III)-OH, His-Fe(III)-SH/His-Fe(II)- Met and final complexes, respectively, formed after addition of Na 2 S. The final complex is an admixture of His-Fe(II)-O 2, His-Fe(III)-OH and modified Fe(III) complexes, or one of the three complexes, depending on the protein (cf. Table 1). Buffer: 50 mM Tris-HCl, pH ,

4 Wavelength (nm) Absorbance 417 nm (1) 424 nm (4) 418 nm (2) 427nm (3) 658 nm 5X Fig. S3. (A) Spectra of R97A without Na 2 S (black: 1), with Na 2 S (blue: 2), with Na 2 S + Na 2 S 2 O 4 (red: 3) and with Na 2 S + Na 2 S 2 O 4 + CO (gray: 4). The spectra correspond with those in Fig. S2 (E).

5 Wavelength (nm) Absorbance 417 nm (1) 424 nm (4) 418 nm (2), (3) 658 nm 5X Fig. S3. (B) Spectra of R97A without Na 2 S (black: 1), with Na 2 S (blue: 2), with CO (red: 3) and with Na 2 S + CO + Na 2 S 2 O 4 (gray: 4). The spectra correspond with those in Fig. S2 (E).

6 m/z C 33 H 31 N 4 O 5 Fe 1 C 33 H 29 N 4 O 4 Fe 1 C 31 H 28 N 4 O 3 Fe 1 C 30 H 25 N 4 O 3 Fe 1 C 30 H 27 N 4 O 1 Fe 1 C 29 H 25 N 4 O 1 Fe 1 C 34 H 32 N 4 O 4 Fe 1 C 32 H 29 N 4 O 2 Fe 1 C 31 H 28 N 4 O 2 Fe 1 C 30 H 26 N 4 Fe 1 C 29 H 25 N 4 Fe 1 C 32 H 27 N 4 O 1 Fe 1 A B Fig. S4: Tandem mass spectra of heme (m/z 616,1763) (A) and verdoheme (m/z ) (B). Calculated elemental composition of individual fragment ions are depicted in grey above each m/z value.

7 Fig. S5: Time-dependent verdoheme formation in R97A (20  M) by adding Na 2 S (4 mM). The MS spectra were recorded at 0, 5, 10, 20, 30, 60, 90, 120 and 180 min of incubation. The numbers on the right at each particular time represent the ratio between the verdoheme and heme signal intensities in the mass spectrum.

8 Wavelength (nm) Absorbance 417 nm (1) 427 nm (3) 418 nm (2) nm (4) 658 nm 5X Fig. S6: (A) Spectra of R97I without Na 2 S (black: 1), with Na 2 S (blue: 2), with Na 2 S + Na 2 S 2 O 4 (red: 3) and with Na 2 S + Na 2 S 2 O 4 + CO (gray: 4). The spectra correspond with those in Fig. S2 (G).

9 Wavelength (nm) Absorbance 417 nm (1) 418 nm (2), (3) nm (4) 658 nm 5X Fig. S6: (B) Spectra of R97I without Na 2 S (black: 1), with Na 2 S (blue: 2), with CO (red: 3) and with Na 2 S + CO + Na 2 S 2 O 4 (gray: 4). The spectra correspond with those in Fig. S2 (G).

10 Fig. S7: Time-dependent verdoheme formation in R97I (20  M) by adding Na 2 S (4 mM). The MS spectra were recorded at 0, 5, 10, 20, 30, 60, 90, 120 and 180 min of incubation. The numbers on the right at each particular time represent the ratio between the verdoheme and heme signal intensities in the mass spectrum.

11 Biliverdin C33H34N4O Heme C34H32N4O4Fe Verdoheme C33H31N4O5Fe Sulfheme C34H32N4O4Fe1S1 Fig. S8. Comparison of the relative intensities of signals of other heme degradation products with the heme/verdoheme signals. Other heme degradation products are much less intense (1% of the base peak – heme/verdoheme – intensity and less) than heme/verdoheme signals. The other non-labeled signals are matrix adducts.


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