Supplementary Data for

Effects of hydrogen sulfide on the heme coordination structure and catalytic activity of the globin-coupled oxygen sensor AfGcHK

Veronika Fojtikova‡1, Martina Bartosova‡1, Petr Man‡,¶, Martin Stranava‡,

Toru Shimizu‡ and Marketa Martinkova‡2

‡Department of Biochemistry, Faculty of Science, Charles University in Prague

Hlavova 2030, Prague 2, 128 43 Czech Republic

¶ Biotechnology and Biomedicine Centre (BioCeV), Institute of Microbiology of the Czech Academy of Science, v.v.i.

Prumyslova 595, Vestec, 252 42 Czech Republic

Table S1: Absorption spectral bands (nm) of AfGcHK, other heme-based oxygen sensors, YddV and Ec DOS, hemoglobin (Hb), myoglobin (Mb), verdoheme and sulfheme.

Heme protein Soret visible references

AfGcHK

Heme Fe(III)-OH 6cLS 411 538 (Kitanishi et al., 2011)

Heme Fe(III)-SH 6cLS 423-426 549-551 570(br) this work

Heme Fe(II) 5cHS 431 559 (Kitanishi et al., 2011)

Heme Fe(II)-O2 6cLS 413 545 580 (Kitanishi et al., 2011)

Heme Fe(II)-CO 6cLS 420 541 565 (Kitanishi et al., 2011)

YddV

Heme Fe(III) 5cHS 392-394 506-509 644-651 (Kitanishi et al., 2010)

Heme Fe(III)-SH 6cLS 421-427 545-550 570(br) this work

Heme Fe(II) 5cHS 430-432 557-560 (Kitanishi et al., 2010)

Heme Fe(II)-O2 6cLS 411-414 542-545 578-579 (Kitanishi et al., 2010)

Heme Fe(II)-CO 6cLS 420 539-540 566-567 (Kitanishi et al., 2010)

Ec DOS

Heme Fe(III)-OH 6cLS 417 530 564 (Hirata et al., 2003)

Heme Fe(III)-SH 6cLS 422-426 541-547 572-580 (Takahashi et al., 2012; Yan et al., 2015)

Heme Fe(II) 6cLS 428-429 532-533 557-563 (Hirata et al., 2003)

Heme Fe(II)-O2 6cLS 416-418 542 578-580 (Hirata et al., 2003)

Heme Fe(II)-CO 6cLS 423 540 570 (Hirata et al., 2003)

Verdoheme Fe(III) 352-399 540 678 (Sakamoto et al., 2000)

Verdoheme Fe(II) 395-399 501-536(br) 648-680(br)(Sakamoto et al.,2000)

Heme Fe(III)-SH (Hb) 423-427 541-547 572-580 (Nicoletti et al., 2010; Ramos-Alvarez et al., 2013)

Sulfheme Fe(III) (Mb) 404 492-593 (br) (Andersson et al., 1984; Berzofsky et al., 1971)

Sulfheme Fe(II) (Mb) 421 508-570 (br) 617 (Andersson et al., 1984; Berzofsky et al., 1971)

Table S2: Changes in the position of the Soret band (nm) and the coordination structure of the heme Fe(III) complexes of the isolated WT heme-binding domain of YddV (YddV-heme) and its Y43 and L65 mutants (5 mM) upon treatment with Na2S (5 mM).

Initial Complex Intermediate Complexes Final Complex

Fe(III) Fe(III)-SH Fe(II) Fe(II)-O2

WT 394 413 432 Fe(II)

5cHS 6cLS 6cLS

Y43A 391 427 438 Degradation 416 nm

5cHS 6cLS

Y43F 393 425 Degradation 420 nm

5cHS 6cLS

Y43W 391 410-412 Degradation 408 nm

5cHS 6cLS

L65G 405 Degradation 421 nm

6cLS

L65M 403 425 Degradation 421 nm

6cLS 6cLS

L65Q 405 421 413 Degradation 421 nm

6cLS 6cLS 6cLS

L65T 404 421 Degradation 422 nm

6cLS 6cLS

Fig. S1 Lineweaver-Burk plots used to determine apparent KmATP and VmaxATP values for autophosphorylation reaction catalyzed by the Fe(III)-OH- form of AfGcHK before Na2S treatment (A), results after 75 seconds’ incubation with Na2S (B), the results after 75 minutes’ incubation with Na2S (C).

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Fig. S2 Changes in the UV-visible spectra of the WT globin-coupled oxygen sensor domain of YddV (YddV-heme) and its Y43 and L65 mutants (5 μM) upon treatment with Na2S (5 mM). (A) Wild type; (B) Y43A; (C) Y43F; (D) Y43W; (E) L65G; (F) L65M; (G) L65Q; (H) L65T. Black line: spectra of the Fe(III) complexes without Na2S; grey line: spectra observed 10 min after the addition of Na2S; dashed black line: final spectra observed 90 minutes after the addition of Na2S.

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Fig. S3 MS spectra of the heme – verdoheme region for WT AfGcHK (A) and its mutants (B-H) after 60 minutes’ incubation with Na2S (5 mM). Isotopic patterns of heme (m/z 616.17674) and verdoheme (m/z 619.16383) are shown and position of the monoisotopic peaks for both heme species is marked with a solid grey line. Verdoheme is formed in the cases of the L68G (F), L68N (G) and L68V (H) mutants.

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