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Accommodation of CO in the di-heme active site of cytochrome bd terminal oxidase from Escherichia coli
Journal of Inorganic Biochemistry 118 (2013) 65–67
Contents lists available at SciVerse ScienceDirect
Journal of Inorganic Biochemistry journal homepage: www.elsevier.com/locate/jinorgbio
Accommodation of CO in the di-heme active site of cytochrome bd terminal oxidase from Escherichia coli☆ Vitaliy B. Borisov a,⁎, Michael I. Verkhovsky b, 1 a b
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119991, Russian Federation Helsinki Bioenergetics Group, Institute of Biotechnology, University of Helsinki, PB 65 (Viikinkaari 1), FI-00014 Helsinki, Finland
a r t i c l e
i n f o
Article history: Received 30 June 2012 Received in revised form 1 September 2012 Accepted 16 September 2012 Available online 24 September 2012 Keywords: Oxygen chemistry Hemeproteins Ligand binding Flash photolysis Reaction mechanisms
a b s t r a c t Catalytic mechanisms of reduction of O2 to 2H2O by respiratory terminal oxidases have been extensively investigated. Tri-heme (b558, b595, d) cytochrome bd oxidases presumably utilize a dihemic site composed of high-spin hemes d and b595. We performed a CO photolysis/recombination study of the purified fully reduced cytochrome bd from Escherichia coli. Spectrum of CO photolysis suggests photodissociation of the ligand from heme d and from part of heme b595. This is the first clear evidence of interaction of heme b595 with CO at room temperature. The amount of the heme d-CO species is higher after recombination than before photolysis. In the enzyme population with heme b595 bound to CO, heme d remains unliganded, hence the dihemic O2-reducing pocket in cytochrome bd can bind one rather than two diatomic molecules. Occupancy of the site by one ligand molecule probably blocks access of a second molecule. Thus cytochrome bd exhibits strong negative cooperativity in ligand binding. Immediately after photolysis/recombination CO occupies 100% of the heme d sites, whereas after equilibration, the ligand gets located at heme d in 90–95% and at heme b595 in 5–10% of the cytochrome. The equilibration process is possibly associated with an exchange of heme d endogenous ligand. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Respiratory terminal oxidases catalyze the reduction of O2 to 2H2O, the reaction that occurs at a bimetallic active site. The binuclear site of heme–copper oxidases which are true proton pumps consists of a heme and a copper ion (CuB). Their reaction mechanism and interactions with small molecules like CO and NO have been intensively studied [1–5]. The tri-heme cytochrome bd oxidases, which refer to iron– chlorine proteins [6–8], contain no copper [9–11] and are thought to bind, activate and reduce O2 using a di-heme active site composed of the high-spin pentacoordinate hemes d and b595 [12–21]. The third heme, low-spin hexacoordinate b558, transfers electrons from the natural electron donor quinol towards the di-heme site [22]. Cytochromes bd can generate proton-motive force but without invoking a ‘proton pump’ mechanism [18,23–28]. They are widely distributed in pathogenic bacteria and responsible for a number of vitally important physiological functions [29–31] which include conferring resistance to NO stress [32–36]. Earlier CO was used as a ligand for probing the cytochrome bd active site [12,14–16,20,37]. The ligand was reported to react to heme d and at high concentrations to a fraction of heme b558 ☆ The respiratory chain of Escherichia coli contains the two bd-type oxidases, bd-I and bd-II. Unless otherwise stated, we refer to the bd-I oxidase throughout the manuscript. ⁎ Corresponding author at: Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskie Gory, Moscow 119991, Russian Federation. Tel.: +7 495 9395149; fax: +7 495 9393181. E-mail address: [email protected] (V.B. Borisov). 1 Deceased 4th October, 2011. 0162-0134/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jinorgbio.2012.09.016
[14,37]. Clear evidence for interaction of CO with a small population of heme b595 was shown only at low temperatures [12,16]. It was speculated that CO partitions between hemes d and b595 in accordance with their ligand intrinsic affinities, the affinity of heme d for CO being much higher than that of heme b595 [14,16]. It is not clear, however, if in the enzyme population in which CO is bound to heme b595, heme d is also in the CO-ligated state, i.e., whether the di-heme active site can accommodate the two diatomic molecules at the same time. CO was purchased from AGA. Cytochrome bd from Escherichia coli strain GO105/pTK1 was purified as described in [37]. The sample contained the CO-inhibited dithionite-reduced enzyme under anaerobic conditions (cytochrome bd (ca. 12 μM) in 60 mM HEPES-KOH/ 25 mM phosphate buffer (pH 7.5), N-lauroyl-sarcosine (0.025%), n-dodecyl-β-D-maltoside (0.1%), 30 μM CO) was kept in the dark prior to the flash. Flash photolysis measurements were carried out at room temperature using a diode array kinetic spectrophotometer made by Unisoku Instruments (Kyoto, Japan). The photolysis was initiated by a xenon camera flash (Braun 2000/320 BVC). A camera shutter (Olympus OM-10) was used to block the probe beam until a few milliseconds before the flash. The sample after photolysis was kept under the monitoring light. The enzyme concentration was determined from the dithionite-reduced-minus-‘as-isolated’ difference absorption spectrum using the value of Δε628−607 = 10.8 mM−1 cm−1 [14]. Data analysis was carried out using MATLAB (The Mathworks, South Natick, MA). Fig. 1A shows the absorption spectrum of photolysis of the CO complex of the fully reduced (R) cytochrome bd from E. coli (thick line). A maximum at 623 nm and a minimum at 643 nm suggest
66
V.B. Borisov, M.I. Verkhovsky / Journal of Inorganic Biochemistry 118 (2013) 65–67
Fig. 1. (A) Spectrum of CO photolysis of cytochrome bd (thick), and difference between spectra before photolysis and after recombination (thin). (B) Thick minus thin. (C) Kinetics at 623 nm.
photodissociation of CO from ferrous heme d [16]. A shoulder at about 600 nm is indicative of CO photolysis from a fraction of ferrous heme b595 [16]. As shown by a selected kinetics at 623 nm (Fig. 1C), following recombination the flash-induced absorption change does not return to the initial level (before flash) but surprisingly goes below zero. This implies that the amount of the heme d-CO species appears to be unexpectedly higher after flash-induced recombination than before photolysis. The difference between the R-CO spectra before photolysis (‘dark’ state) and after recombination (‘light’ state) reveals a maximum at 623 nm and a minimum at 643 nm (Fig. 1A, thin line) that supports such a conclusion. A normalization factor of ~ 10 is required for that difference spectrum to match the spectrum
of photolysis by the peak-to-trough intensity. Fig. 1B demonstrates the difference between the two spectra (thick minus thin) shown in Fig. 1A. The difference shows the two clear spectral features, a maximum at about 595 nm and a minimum around 630 nm. The 595 nm maximum undoubtedly reflects the formation of the unliganded ferrous heme b595 after photodissociation of CO [16]. This is the first unambiguous evidence of the interaction of heme b595 (5–10% in different experiments) with CO at room temperature. A trough at about 630 nm may indicate a change in the heme d coordination sphere induced by flash. Furthermore, the data suggest that in the enzyme population with heme b595 bound to CO, heme d remains unliganded. Thus, one may conclude that the di-heme oxygen reducing pocket in cytochrome bd can bind one rather than two diatomic molecules like CO (Fig. 2). This can hardly be explained by ordinary steric restrictions within the pocket because the C\O bond length is only ~ 0.11 nm, whereas an estimate of the Fe-to-Fe distance between heme d and heme b595 was reported to be ~ 1 nm [19]. A possible explanation is that occupancy of the di-heme site by one ligand molecule blocks access of a second ligand molecule to the site. Thus the cytochrome bd di-heme active site exhibits strong negative cooperativity in ligand binding. Immediately after photolysis/recombination in the sample kept under the monitoring light CO occupies 100% of the heme d sites. Then under dark conditions the ligand equilibrates between the two hemes in accordance with their intrinsic affinities for CO, heme d having a higher affinity. After the equilibration, the ligand is located at heme d in 90–95% of the cytochrome and at heme b595 in 5–10% of the enzyme (Fig. 2). The equilibration process may be associated with an exchange of an endogenous ligand in the coordination sphere of heme d, as evidenced by the 630 nm trough (Fig. 1B). The latter assumption is in line with an earlier report that reduction of cytochrome bd is linked to binding or exchange of an endogenous protein ligand to heme d [38]. It is worth noting that well-documented cases of negative cooperativity are rare [39]. It can be speculated that in vivo the cytochrome bd population, in which heme b595 can potentially bind a small molecule like CO, might be implicated in an alternative physiological function so far unrevealed. In conclusion, we have shown that when the R cytochrome bd is complexed with CO at room temperature, its di-heme (d/b595) active site accommodates only one ligand molecule: CO is bound to heme d in 90–95% of the enzyme and to heme b595 — in 5–10% of the cytochrome. This study provides further insight into the molecular mechanisms of functioning of terminal oxidases and other enzymes that contain a bimetallic active site.
Fig. 2. Model for CO binding to proposed di-heme active site of cytochrome bd.
V.B. Borisov, M.I. Verkhovsky / Journal of Inorganic Biochemistry 118 (2013) 65–67
Acknowledgments We thank Prof. R. B. Gennis (Urbana, USA) for the strain of E. coli GO105/pTK1 and Dr. I. Belevich (Helsinki, Finland) for his assistance with the preparation of the graphical abstract. This work was supported by the Russian Foundation for Basic Research, grant 11-04-00031-a (to V.B.B.), and the Biocentrum Helsinki, Sigrid Jusélius Foundation, the Academy of Finland (to M.I.V.). References [1] J.P. Collman, R. Boulatov, C.J. Sunderland, L. Fu, Chem. Rev. 104 (2004) 561–588. [2] P. Sarti, E. Forte, D. Mastronicola, A. Giuffrè, M. Arese, Biochim. Biophys. Acta 1817 (2012) 610–619. [3] G.C. Brown, C.E. Cooper, FEBS Lett. 356 (1994) 295–298. [4] S. Brown, J.N. Rumbley, A.J. Moody, J.W. Thomas, R.B. Gennis, P.R. Rich, Biochim. Biophys. Acta 1183 (1994) 521–532. [5] I.A. Smirnova, D. Zaslavsky, J.A. Fee, R.B. Gennis, P. Brzezinski, J. Bioenerg. Biomembr. 40 (2008) 281–287. [6] J.H. Dawson, A.M. Bracete, A.M. Huff, S. Kadkhodayan, C.M. Zeitler, M. Sono, C.K. Chang, P.C. Loewen, FEBS Lett. 295 (1991) 123–126. [7] M. Sono, A.M. Bracete, A.M. Huff, M. Ikeda-Saito, J.H. Dawson, Proc. Natl. Acad. Sci. U. S. A. 88 (1991) 11148–11152. [8] E.D. Coulter, J. Cheek, A.P. Ledbetter, C.K. Chang, J.H. Dawson, Biochem. Biophys. Res. Commun. 279 (2000) 1011–1015. [9] T. Mogi, M. Tsubaki, H. Hori, H. Miyoshi, H. Nakamura, Y. Anraku, J. Biochem. Mol. Biol. Biophys. 2 (1998) 79–110. [10] R.K. Poole, G.M. Cook, Adv. Microb. Physiol. 43 (2000) 165–224. [11] V.B. Borisov, R.B. Gennis, J. Hemp, M.I. Verkhovsky, Biochim. Biophys. Acta 1807 (2011) 1398–1413. [12] J.J. Hill, J.O. Alben, R.B. Gennis, Proc. Natl. Acad. Sci. U. S. A. 90 (1993) 5863–5867. [13] M. Tsubaki, H. Hori, T. Mogi, Y. Anraku, J. Biol. Chem. 270 (1995) 28565–28569. [14] V. Borisov, A.M. Arutyunyan, J.P. Osborne, R.B. Gennis, A.A. Konstantinov, Biochemistry 38 (1999) 740–750. [15] M.H. Vos, V.B. Borisov, U. Liebl, J.-L. Martin, A.A. Konstantinov, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 1554–1559. [16] V.B. Borisov, S.E. Sedelnikova, R.K. Poole, A.A. Konstantinov, J. Biol. Chem. 276 (2001) 22095–22099. [17] V.B. Borisov, U. Liebl, F. Rappaport, J.-L. Martin, J. Zhang, R.B. Gennis, A.A. Konstantinov, M.H. Vos, Biochemistry 41 (2002) 1654–1662.
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[18] I. Belevich, V.B. Borisov, J. Zhang, K. Yang, A.A. Konstantinov, R.B. Gennis, M.I. Verkhovsky, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 3657–3662. [19] A.M. Arutyunyan, V.B. Borisov, V.I. Novoderezhkin, J. Ghaim, J. Zhang, R.B. Gennis, A.A. Konstantinov, Biochemistry 47 (2008) 1752–1759. [20] F. Rappaport, J. Zhang, M.H. Vos, R.B. Gennis, V.B. Borisov, Biochim. Biophys. Acta 1797 (2010) 1657–1664. [21] A.M. Arutyunyan, J. Sakamoto, M. Inadome, Y. Kabashima, V.B. Borisov, Biochim. Biophys. Acta 1817 (2012) 2087–2094. [22] A. Hata-Tanaka, K. Matsuura, S. Itoh, Y. Anraku, Biochim. Biophys. Acta 893 (1987) 289–295. [23] A. Puustinen, M. Finel, T. Haltia, R.B. Gennis, M. Wikström, Biochemistry 30 (1991) 3936–3942. [24] A. Jasaitis, V.B. Borisov, N.P. Belevich, J.E. Morgan, A.A. Konstantinov, M.I. Verkhovsky, Biochemistry 39 (2000) 13800–13809. [25] I. Belevich, V.B. Borisov, M.I. Verkhovsky, J. Biol. Chem. 282 (2007) 28514–28519. [26] V.B. Borisov, I. Belevich, D.A. Bloch, T. Mogi, M.I. Verkhovsky, Biochemistry 47 (2008) 7907–7914. [27] J.F. Kolonay Jr., R.J. Maier, J. Bacteriol. 179 (1997) 3813–3817. [28] V.B. Borisov, R. Murali, M.L. Verkhovskaya, D.A. Bloch, H. Han, R.B. Gennis, M.I. Verkhovsky, Proc. Natl. Acad. Sci. U. S. A. 108 (2011) 17320–17324. [29] A. Giuffrè, V.B. Borisov, D. Mastronicola, P. Sarti, E. Forte, FEBS Lett. 586 (2012) 622–629. [30] E. Forte, V.B. Borisov, A.A. Konstantinov, M. Brunori, A. Giuffrè, P. Sarti, Ital. J. Biochem. 56 (2007) 265–269. [31] L. Winstedt, L. Frankenberg, L. Hederstedt, C. von Wachenfeldt, J. Bacteriol. 182 (2000) 3863–3866. [32] V.B. Borisov, E. Forte, A.A. Konstantinov, R.K. Poole, P. Sarti, A. Giuffrè, FEBS Lett. 576 (2004) 201–204. [33] V.B. Borisov, E. Forte, P. Sarti, M. Brunori, A.A. Konstantinov, A. Giuffrè, FEBS Lett. 580 (2006) 4823–4826. [34] V.B. Borisov, E. Forte, P. Sarti, M. Brunori, A.A. Konstantinov, A. Giuffrè, Biochem. Biophys. Res. Commun. 355 (2007) 97–102. [35] M.G. Mason, M. Shepherd, P. Nicholls, P.S. Dobbin, K.S. Dodsworth, R.K. Poole, C.E. Cooper, Nat. Chem. Biol. 5 (2009) 94–96. [36] V.B. Borisov, E. Forte, A. Giuffrè, A. Konstantinov, P. Sarti, J. Inorg. Biochem. 103 (2009) 1185–1187. [37] V.B. Borisov, Biochem. Mosc. 73 (2008) 14–22 (translated from Biokhimiya (in Russian) (2008), 73, 18–28). [38] N. Azarkina, V. Borisov, A.A. Konstantinov, FEBS Lett. 416 (1997) 171–174. [39] D.L. Nelson, M.M. Cox, Lehninger Principles of Biochemistry, Fourth ed. W.H. Freeman, New York, 2004.
Contents lists available at SciVerse ScienceDirect
Journal of Inorganic Biochemistry journal homepage: www.elsevier.com/locate/jinorgbio
Accommodation of CO in the di-heme active site of cytochrome bd terminal oxidase from Escherichia coli☆ Vitaliy B. Borisov a,⁎, Michael I. Verkhovsky b, 1 a b
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119991, Russian Federation Helsinki Bioenergetics Group, Institute of Biotechnology, University of Helsinki, PB 65 (Viikinkaari 1), FI-00014 Helsinki, Finland
a r t i c l e
i n f o
Article history: Received 30 June 2012 Received in revised form 1 September 2012 Accepted 16 September 2012 Available online 24 September 2012 Keywords: Oxygen chemistry Hemeproteins Ligand binding Flash photolysis Reaction mechanisms
a b s t r a c t Catalytic mechanisms of reduction of O2 to 2H2O by respiratory terminal oxidases have been extensively investigated. Tri-heme (b558, b595, d) cytochrome bd oxidases presumably utilize a dihemic site composed of high-spin hemes d and b595. We performed a CO photolysis/recombination study of the purified fully reduced cytochrome bd from Escherichia coli. Spectrum of CO photolysis suggests photodissociation of the ligand from heme d and from part of heme b595. This is the first clear evidence of interaction of heme b595 with CO at room temperature. The amount of the heme d-CO species is higher after recombination than before photolysis. In the enzyme population with heme b595 bound to CO, heme d remains unliganded, hence the dihemic O2-reducing pocket in cytochrome bd can bind one rather than two diatomic molecules. Occupancy of the site by one ligand molecule probably blocks access of a second molecule. Thus cytochrome bd exhibits strong negative cooperativity in ligand binding. Immediately after photolysis/recombination CO occupies 100% of the heme d sites, whereas after equilibration, the ligand gets located at heme d in 90–95% and at heme b595 in 5–10% of the cytochrome. The equilibration process is possibly associated with an exchange of heme d endogenous ligand. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Respiratory terminal oxidases catalyze the reduction of O2 to 2H2O, the reaction that occurs at a bimetallic active site. The binuclear site of heme–copper oxidases which are true proton pumps consists of a heme and a copper ion (CuB). Their reaction mechanism and interactions with small molecules like CO and NO have been intensively studied [1–5]. The tri-heme cytochrome bd oxidases, which refer to iron– chlorine proteins [6–8], contain no copper [9–11] and are thought to bind, activate and reduce O2 using a di-heme active site composed of the high-spin pentacoordinate hemes d and b595 [12–21]. The third heme, low-spin hexacoordinate b558, transfers electrons from the natural electron donor quinol towards the di-heme site [22]. Cytochromes bd can generate proton-motive force but without invoking a ‘proton pump’ mechanism [18,23–28]. They are widely distributed in pathogenic bacteria and responsible for a number of vitally important physiological functions [29–31] which include conferring resistance to NO stress [32–36]. Earlier CO was used as a ligand for probing the cytochrome bd active site [12,14–16,20,37]. The ligand was reported to react to heme d and at high concentrations to a fraction of heme b558 ☆ The respiratory chain of Escherichia coli contains the two bd-type oxidases, bd-I and bd-II. Unless otherwise stated, we refer to the bd-I oxidase throughout the manuscript. ⁎ Corresponding author at: Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskie Gory, Moscow 119991, Russian Federation. Tel.: +7 495 9395149; fax: +7 495 9393181. E-mail address: [email protected] (V.B. Borisov). 1 Deceased 4th October, 2011. 0162-0134/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jinorgbio.2012.09.016
[14,37]. Clear evidence for interaction of CO with a small population of heme b595 was shown only at low temperatures [12,16]. It was speculated that CO partitions between hemes d and b595 in accordance with their ligand intrinsic affinities, the affinity of heme d for CO being much higher than that of heme b595 [14,16]. It is not clear, however, if in the enzyme population in which CO is bound to heme b595, heme d is also in the CO-ligated state, i.e., whether the di-heme active site can accommodate the two diatomic molecules at the same time. CO was purchased from AGA. Cytochrome bd from Escherichia coli strain GO105/pTK1 was purified as described in [37]. The sample contained the CO-inhibited dithionite-reduced enzyme under anaerobic conditions (cytochrome bd (ca. 12 μM) in 60 mM HEPES-KOH/ 25 mM phosphate buffer (pH 7.5), N-lauroyl-sarcosine (0.025%), n-dodecyl-β-D-maltoside (0.1%), 30 μM CO) was kept in the dark prior to the flash. Flash photolysis measurements were carried out at room temperature using a diode array kinetic spectrophotometer made by Unisoku Instruments (Kyoto, Japan). The photolysis was initiated by a xenon camera flash (Braun 2000/320 BVC). A camera shutter (Olympus OM-10) was used to block the probe beam until a few milliseconds before the flash. The sample after photolysis was kept under the monitoring light. The enzyme concentration was determined from the dithionite-reduced-minus-‘as-isolated’ difference absorption spectrum using the value of Δε628−607 = 10.8 mM−1 cm−1 [14]. Data analysis was carried out using MATLAB (The Mathworks, South Natick, MA). Fig. 1A shows the absorption spectrum of photolysis of the CO complex of the fully reduced (R) cytochrome bd from E. coli (thick line). A maximum at 623 nm and a minimum at 643 nm suggest
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V.B. Borisov, M.I. Verkhovsky / Journal of Inorganic Biochemistry 118 (2013) 65–67
Fig. 1. (A) Spectrum of CO photolysis of cytochrome bd (thick), and difference between spectra before photolysis and after recombination (thin). (B) Thick minus thin. (C) Kinetics at 623 nm.
photodissociation of CO from ferrous heme d [16]. A shoulder at about 600 nm is indicative of CO photolysis from a fraction of ferrous heme b595 [16]. As shown by a selected kinetics at 623 nm (Fig. 1C), following recombination the flash-induced absorption change does not return to the initial level (before flash) but surprisingly goes below zero. This implies that the amount of the heme d-CO species appears to be unexpectedly higher after flash-induced recombination than before photolysis. The difference between the R-CO spectra before photolysis (‘dark’ state) and after recombination (‘light’ state) reveals a maximum at 623 nm and a minimum at 643 nm (Fig. 1A, thin line) that supports such a conclusion. A normalization factor of ~ 10 is required for that difference spectrum to match the spectrum
of photolysis by the peak-to-trough intensity. Fig. 1B demonstrates the difference between the two spectra (thick minus thin) shown in Fig. 1A. The difference shows the two clear spectral features, a maximum at about 595 nm and a minimum around 630 nm. The 595 nm maximum undoubtedly reflects the formation of the unliganded ferrous heme b595 after photodissociation of CO [16]. This is the first unambiguous evidence of the interaction of heme b595 (5–10% in different experiments) with CO at room temperature. A trough at about 630 nm may indicate a change in the heme d coordination sphere induced by flash. Furthermore, the data suggest that in the enzyme population with heme b595 bound to CO, heme d remains unliganded. Thus, one may conclude that the di-heme oxygen reducing pocket in cytochrome bd can bind one rather than two diatomic molecules like CO (Fig. 2). This can hardly be explained by ordinary steric restrictions within the pocket because the C\O bond length is only ~ 0.11 nm, whereas an estimate of the Fe-to-Fe distance between heme d and heme b595 was reported to be ~ 1 nm [19]. A possible explanation is that occupancy of the di-heme site by one ligand molecule blocks access of a second ligand molecule to the site. Thus the cytochrome bd di-heme active site exhibits strong negative cooperativity in ligand binding. Immediately after photolysis/recombination in the sample kept under the monitoring light CO occupies 100% of the heme d sites. Then under dark conditions the ligand equilibrates between the two hemes in accordance with their intrinsic affinities for CO, heme d having a higher affinity. After the equilibration, the ligand is located at heme d in 90–95% of the cytochrome and at heme b595 in 5–10% of the enzyme (Fig. 2). The equilibration process may be associated with an exchange of an endogenous ligand in the coordination sphere of heme d, as evidenced by the 630 nm trough (Fig. 1B). The latter assumption is in line with an earlier report that reduction of cytochrome bd is linked to binding or exchange of an endogenous protein ligand to heme d [38]. It is worth noting that well-documented cases of negative cooperativity are rare [39]. It can be speculated that in vivo the cytochrome bd population, in which heme b595 can potentially bind a small molecule like CO, might be implicated in an alternative physiological function so far unrevealed. In conclusion, we have shown that when the R cytochrome bd is complexed with CO at room temperature, its di-heme (d/b595) active site accommodates only one ligand molecule: CO is bound to heme d in 90–95% of the enzyme and to heme b595 — in 5–10% of the cytochrome. This study provides further insight into the molecular mechanisms of functioning of terminal oxidases and other enzymes that contain a bimetallic active site.
Fig. 2. Model for CO binding to proposed di-heme active site of cytochrome bd.
V.B. Borisov, M.I. Verkhovsky / Journal of Inorganic Biochemistry 118 (2013) 65–67
Acknowledgments We thank Prof. R. B. Gennis (Urbana, USA) for the strain of E. coli GO105/pTK1 and Dr. I. Belevich (Helsinki, Finland) for his assistance with the preparation of the graphical abstract. This work was supported by the Russian Foundation for Basic Research, grant 11-04-00031-a (to V.B.B.), and the Biocentrum Helsinki, Sigrid Jusélius Foundation, the Academy of Finland (to M.I.V.). References [1] J.P. Collman, R. Boulatov, C.J. Sunderland, L. Fu, Chem. Rev. 104 (2004) 561–588. [2] P. Sarti, E. Forte, D. Mastronicola, A. Giuffrè, M. Arese, Biochim. Biophys. Acta 1817 (2012) 610–619. [3] G.C. Brown, C.E. Cooper, FEBS Lett. 356 (1994) 295–298. [4] S. Brown, J.N. Rumbley, A.J. Moody, J.W. Thomas, R.B. Gennis, P.R. Rich, Biochim. Biophys. Acta 1183 (1994) 521–532. [5] I.A. Smirnova, D. Zaslavsky, J.A. Fee, R.B. Gennis, P. Brzezinski, J. Bioenerg. Biomembr. 40 (2008) 281–287. [6] J.H. Dawson, A.M. Bracete, A.M. Huff, S. Kadkhodayan, C.M. Zeitler, M. Sono, C.K. Chang, P.C. Loewen, FEBS Lett. 295 (1991) 123–126. [7] M. Sono, A.M. Bracete, A.M. Huff, M. Ikeda-Saito, J.H. Dawson, Proc. Natl. Acad. Sci. U. S. A. 88 (1991) 11148–11152. [8] E.D. Coulter, J. Cheek, A.P. Ledbetter, C.K. Chang, J.H. Dawson, Biochem. Biophys. Res. Commun. 279 (2000) 1011–1015. [9] T. Mogi, M. Tsubaki, H. Hori, H. Miyoshi, H. Nakamura, Y. Anraku, J. Biochem. Mol. Biol. Biophys. 2 (1998) 79–110. [10] R.K. Poole, G.M. Cook, Adv. Microb. Physiol. 43 (2000) 165–224. [11] V.B. Borisov, R.B. Gennis, J. Hemp, M.I. Verkhovsky, Biochim. Biophys. Acta 1807 (2011) 1398–1413. [12] J.J. Hill, J.O. Alben, R.B. Gennis, Proc. Natl. Acad. Sci. U. S. A. 90 (1993) 5863–5867. [13] M. Tsubaki, H. Hori, T. Mogi, Y. Anraku, J. Biol. Chem. 270 (1995) 28565–28569. [14] V. Borisov, A.M. Arutyunyan, J.P. Osborne, R.B. Gennis, A.A. Konstantinov, Biochemistry 38 (1999) 740–750. [15] M.H. Vos, V.B. Borisov, U. Liebl, J.-L. Martin, A.A. Konstantinov, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 1554–1559. [16] V.B. Borisov, S.E. Sedelnikova, R.K. Poole, A.A. Konstantinov, J. Biol. Chem. 276 (2001) 22095–22099. [17] V.B. Borisov, U. Liebl, F. Rappaport, J.-L. Martin, J. Zhang, R.B. Gennis, A.A. Konstantinov, M.H. Vos, Biochemistry 41 (2002) 1654–1662.
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