20KD

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

20KDEc DOS-PAS

12KD

WT

M95

AM

95H

M95

L

R97I

R97A

R97E

Mar

ker

x10

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Wavelength (nm)

Abso

rban

ce

A

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Wavelength (nm)

Abso

rban

ce

B

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Wavelength (nm)

Abso

rban

ce

C

Fig. S2

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0.20

0.30

Wavelength (nm)

Abso

rban

ce

D

x5

x5

x5

417 417(418)

424

414418(417)

426

415

416425

x5

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Wavelength (nm)

Abso

rban

ce

E

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Abso

rban

ce

F

G

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Wavelength (nm)

Abso

rban

ce

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 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.

417418

426

533, 562

658

417

417418

658

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Wavelength (nm)

Abso

rban

ce

417 nm (1)

424 nm (4)

418 nm (2)

427nm (3)

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).

300 600500 7004000.00

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Wavelength (nm)

Abso

rban

ce

417 nm (1)

424 nm (4)

418 nm (2), (3) 658 nm

5X

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).

485.1420498.1499

539.1525544.1553

557.1630

616.1763

501.1368

515.1525

545.1267

560.1450

601.1527

619.1632

460 480 500 520 540 560 580 600 m/z

C33H31N4O5Fe1

C33H29N4O4Fe1

C31H28N4O3Fe1

C30H25N4O3Fe1

C30H27N4O1Fe1

C29H25N4O1Fe1

C34H32N4O4Fe1

C32H29N4O2Fe1

C31H28N4O2Fe1C30H26N4Fe1C29H25N4Fe1 C32H27N4O1Fe1

A

B

Fig. S4: Tandem mass spectra of heme (m/z 616,1763) (A) and verdoheme (m/z 619.1632) (B). Calculated elemental composition of individual fragment ions are depicted in grey above each m/z value.

Fig. S5: Time-dependent verdoheme formation in R97A (20 M) 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.

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rban

ce

417 nm (1)

427 nm (3)

418 nm (2)

0.15

424 nm (4)

658 nm5X

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).

300 600500 7004000.00

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Wavelength (nm)

Abso

rban

ce

417 nm (1)

418 nm (2), (3)

0.15

424nm (4)

658 nm5X

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).

Fig. S7: Time-dependent verdoheme formation in R97I (20 M) 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.

619.1643616.1774

583.2551

583.2551BiliverdinC33H34N4O6

616.1767HemeC34H32N4O4Fe1

619.1638VerdohemeC33H31N4O5Fe1

648.1488SulfhemeC34H32N4O4Fe1S1

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.