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

2. 300
600
500
700
400
0.00
0.05
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Wavelength (nm)
Absorbance
300
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700
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Wavelength (nm)
Absorbance
414
418(417) B A
417
417(418)
426
424 x5 x5
x10
300
600
500
700
400
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0.10
0.20
0.30
Wavelength (nm)
Absorbance
300
600
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Wavelength (nm)
Absorbance D C
415
416
425 x5
Fig. S2

3. 300
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Wavelength (nm)
Absorbance
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Wavelength (nm)
Absorbance
417 E F
417
418
426
533, 562 x5
658
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Wavelength (nm)
Absorbance
Fig. S2. Selected spectra of 1 mM wild-type Ec DOS-PAS (A), M95A (B), M95H (C), M95L (D), R97A (E), R97E (F) and R97I (G) proteins formed by adding 200 mM Na2S 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 Na2S. The final complex is an admixture of His-Fe(II)-O2, 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 7.5. G
417
418 x5
658

4. 300 600 500 700 400 0.00 0.05 0.10 Wavelength (nm) Absorbance 658 nm5X
Fig. S3. (A) Spectra of R97A without Na2S (black: 1), with Na2S (blue: 2), with Na2S + Na2S2O4 (red: 3) and with Na2S + Na2S2O4 + CO (gray: 4). The spectra correspond with those in Fig. S2 (E).

5. 300 600 500 700 400 0.00 0.05 0.10 Wavelength (nm) Absorbance 658 nm5X
Fig. S3. (B) Spectra of R97A without Na2S (black: 1), with Na2S (blue: 2), with CO (red: 3) and with Na2S + CO + Na2S2O4 (gray: 4). The spectra correspond with those in Fig. S2 (E).

6. C32H29N4O2Fe1
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. A
C34H32N4O4Fe1
C31H28N4O2Fe1
C30H26N4Fe1
C29H25N4Fe1
C32H27N4O1Fe1
C31H28N4O3Fe1 B
C29H25N4O1Fe1
C33H31N4O5Fe1
C30H27N4O1Fe1
C33H29N4O4Fe1
C30H25N4O3Fe1
460
480
500
520
540
560
580
600
m/z

7. Fig. S5: Time-dependent verdoheme formation in R97A (20 mM) by adding Na2S (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. 300 600 500 700 400 0.00 0.05 0.10 Wavelength (nm) Absorbance 0.155X
Fig. S6: (A) Spectra of R97I without Na2S (black: 1), with Na2S (blue: 2), with Na2S + Na2S2O4 (red: 3) and with Na2S + Na2S2O4 + CO (gray: 4). The spectra correspond with those in Fig. S2 (G).

9. 300 600 500 700 400 0.00 0.05 0.10 Wavelength (nm) Absorbance 0.155X
Fig. S6: (B) Spectra of R97I without Na2S (black: 1), with Na2S (blue: 2), with CO (red: 3) and with Na2S + CO + Na2S2O4 (gray: 4). The spectra correspond with those in Fig. S2 (G).

10. Fig. S7: Time-dependent verdoheme formation in R97I (20 mM) by adding Na2S (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
C33H34N4O6
Heme
C34H32N4O4Fe1
Verdoheme
C33H31N4O5Fe1
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.