Complexation of CcmB with CcmACD safeguards heme translocation for cytochrome c maturation

Abstract

Cytochrome c maturation (CCM), a posttranslational modification involving covalent attachment of heme to polypeptides (apocyt c), is essential for the activity and cellular function of cytochromes c. Here, we identify and substantiate CcmB as heme translocase in bacteria. When in excess, CcmB expels intracellular heme into the periplasm and thus is detrimental to the cell. We then show that complexation with CcmACD ensures heme translocated by CcmB to be used for CCM only. Moreover, structural analysis and atomistic molecular dynamics simulations reveal that CcmB absorbs heme from the membrane to a heme pocket formed in the dimer interface of the transmembrane helix-bundles. These data, collectively by providing detailed insights into the conformational landscape of CcmB during heme entry, fill in the missing link in our understanding of the heme translocation for CCM.

Keywords

CcmABCD / cytochrome c / cytochrome c biosynthesis / heme / heme translocation

References

[1] Shimizu T, Lengalova A, Martínek V, Martínková M. Heme: emergent roles of heme in signal transduction, functional regulation and as catalytic centres. Chem Soc Rev. 2019; 48:5624–5657.

[2] Li Y, Han S, Gao H. Heme homeostasis and its regulation by hemoproteins in bacteria. mLife. 2024; 3:327–342.

[3] Kranz RG, Richard-Fogal C, Taylor JS, Frawley ER. Cytochrome c biogenesis: mechanisms for covalent modifications and trafficking of heme and for heme-iron redox control. Microbiol Mol Biol Rev. 2009; 73:510–528.

[4] Fu H, Jin M, Wan F, Gao H. Shewanella oneidensis cytochrome c maturation component CcmI is essential for heme attachment at the non-canonical motif of nitrite reductase NrfA. Mol Microbiol. 2015; 95:410–425.

[5] Feissner RE, Richard-Fogal CL, Frawley ER, Kranz RG. ABC transporter-mediated release of a haem chaperone allows cytochrome c biogenesis. Mol Microbiol. 2006; 61:219–231.

[6] Lee D, Pervushin K, Bischof D, Braun M, Thöny-Meyer L. Unusual heme–histidine bond in the active site of a chaperone. J Am Chem Soc. 2005; 127:3716–3717.

[7] Harvat EM, Redfield C, Stevens JM, Ferguson SJ. Probing the heme-binding site of the cytochrome c maturation protein CcmE. Biochemistry. 2009; 48:1820–1828.

[8] Gao M, Nakajima An D, Parks JM, Skolnick J. AF2Complex predicts direct physical interactions in multimeric proteins with deep learning. Nat Commun. 2022; 13:1744.

[9] Li J, Zheng W, Gu M, Han L, Luo Y, Yu K, et al. Structures of the CcmABCD heme release complex at multiple states. Nat Commun. 2022; 13:6422.

[10] Ilcu L, Denkhaus L, Brausemann A, Zhang L, Einsle O. Architecture of the heme-translocating CcmABCD/E complex required for cytochrome c maturation. Nat Commun. 2023; 14:5190.

[11] Schulz H, Fabianek RA, Pellicioli EC, Hennecke H, Thöny-Meyer L. Heme transfer to the heme chaperone CcmE during cytochrome c maturation requires the CcmC protein, which may function independently of the ABC-transporter CcmAB. Proc Natl Acad Sci USA. 1999; 96:6462–6467.

[12] Mendez DL, Lowder EP, Tillman DE, Sutherland MC, Collier AL, Rau MJ, et al. Cryo-EM of CcsBA reveals the basis for cytochrome c biogenesis and heme transport. Nat Chem Biol. 2022; 18:101–108.

[13] Richard-Fogal CL, Frawley ER, Kranz RG. Topology and function of CcmD in cytochrome c maturation. J Bacteriol. 2008; 190:3489–3493.

[14] Christensen O, Harvat EM, Thöny-Meyer L, Ferguson SJ, Stevens JM. Loss of ATP hydrolysis activity by CcmAB results in loss of c-type cytochrome synthesis and incomplete processing of CcmE. FEBS J. 2007; 274:2322–2332.

[15] Meng Q, Sun Y, Gao H. Cytochromes c constitute a layer of protection against nitric oxide but not nitrite. Appl Environ Microbiol. 2018; 84:e0125501218.

[16] Guo K, Wang W, Wang H, Lu Z, Gao H. Complex oxidation of apocytochromes c during bacterial cytochrome c maturation. Appl Environ Microbiol. 2019; 85:e0198901919.

[17] Guo K, Feng X, Sun W, Han S, Wu S, Gao H. NapB restores cytochrome c biosynthesis in bacterial dsbD-deficient mutants. Commun Biol. 2022; 5:87.

[18] Feng X, Sun W, Kong L, Gao H. Distinct roles of Shewanella oneidensis thioredoxin in regulation of cellular responses to hydrogen and organic peroxides. Appl Environ Microbiol. 2019; 85:e01700.

[19] Wang W, Wang J, Feng X, Gao H. A common target of nitrite and nitric oxide for respiration inhibition in bacteria. Int J Mol Sci. 2022; 23:13841.

[20] Dong Z, Guo S, Fu H, Gao H. Investigation of a spontaneous mutant reveals novel features of iron uptake in Shewanella oneidensis. Sci Rep. 2017; 7:11788.

[21] Han S, Guo K, Wang W, Tao YJ, Gao H. Bacterial TANGO2 homologs are heme-trafficking proteins that facilitate biosynthesis of cytochromes c. mBio. 2023; 14:01320–01323.

[22] Fu H, Chen H, Wang J, Zhou G, Zhang H, Zhang L, et al. Crp-dependent cytochrome bd oxidase confers nitrite resistance to Shewanella oneidensis. Environ Microbiol. 2013; 15:2198–2212.

[23] Jiang Y, Dong Y, Luo Q, Li N, Wu G, Gao H. Protection from oxidative stress relies mainly on derepression of OxyR-dependent KatB and Dps in Shewanella oneidensis. J Bacteriol. 2014; 196:445–458.

[24] Yin J, Meng Q, Fu H, Gao H. Reduced expression of cytochrome oxidases largely explains cAMP inhibition of aerobic growth in Shewanella oneidensis. Sci Rep. 2016; 6:24449.

[25] Liu L, Feng X, Wang W, Chen Y, Chen Z, Gao H. Free rather than total iron content is critically linked to the Fur physiology in Shewanella oneidensis. Front Microbiol. 2020; 11:593246.

[26] Hopp M-T, Schmalohr BF, Kühl T, Detzel MS, Wißbrock A, Imhof D. Heme determination and quantification methods and their suitability for practical applications and everyday use. Anal Chem. 2020; 92:9429–9440.

[27] Rodriguez EA, Tran GN, Gross LA, Crisp JL, Shu X, Lin JY, et al. A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein. Nat Methods. 2016; 13:763–769.

[28] Beckman DL, Trawick DR, Kranz RG. Bacterial cytochromes c biogenesis. Genes Dev. 1992; 6:268–283.

[29] Richard-Fogal C, Kranz RG. The CcmC:Heme:CcmE complex in heme trafficking and cytochrome c biosynthesis. J Mol Biol. 2010; 401:350–362.

[30] Oberg N, Zallot R, Gerlt JA. EFI-EST, EFI-GNT, and EFI-CGFP: enzyme function initiative EFI. web resource for genomic enzymology tools. J Mol Biol. 2023; 435:168018.

[31] Wakeman CA, Hammer ND, Stauff DL, Attia AS, Anzaldi LL, Dikalov SI, et al. Menaquinone biosynthesis potentiates haem toxicity in Staphylococcus aureus. Mol Microbiol. 2012; 86:1376–1392.

[32] Joubert L, Derré-Bobillot A, Gaudu P, Gruss A, Lechardeur D. HrtBA and menaquinones control haem homeostasis in Lactococcus lactis. Mol Microbiol. 2014; 93:823–833.

[33] Choby JE, Skaar EP. 2016. Heme synthesis and acquisition in bacterial pathogens. J Mol Biol. 2014; 428:3408–3428.

[34] Nakamura H, Hisano T, Rahman MM, Tosha T, Shirouzu M, Shiro Y. Structural basis for heme detoxification by an ATP-binding cassette–type efflux pump in gram-positive pathogenic bacteria. Proc Natl Acad Sci USA. 2022; 119:e2123385119.

[35] Torres AG, Payne SM. Haem iron-transport system in enterohaemorrhagic Escherichia coli O157:H7. Mol Microbiol. 1997; 23:825–833.

[36] Light WR, Olson JS. Transmembrane movement of heme. J Biol Chem. 1990; 265:15623–15631.

[37] Wu D, Mehdipour AR, Finke F, Goojani HG, Groh RR, Grund TN, et al. Dissecting the conformational complexity and mechanism of a bacterial heme transporter. Nat Chem Biol. 2023; 19:992–1003.

[38] Goldman BS, Kranz RG. ABC transporters associated with cytochrome c biogenesis. Res Microbiol. 2001; 152:323–329.

[39] Schulz H, Hennecke H, Thöny-Meyer L. Prototype of a heme chaperone essential for cytochrome c maturation. Science. 1998; 281:1197–1200.

[40] Torres VJ, Stauff DL, Pishchany G, Bezbradica JS, Gordy LE, Iturregui J, et al. A Staphylococcus aureus regulatory system that responds to host heme and modulates virulence. Cell Host Microbe. 2007; 1:109–119.

[41] Bibb LA, Schmitt MP. The ABC transporter HrtAB confers resistance to hemin toxicity and is regulated in a hemin-dependent manner by the ChrAS two-component system in Corynebacterium diphtheriae. J Bacteriol. 2010; 192:4606–4617.

[42] Lechardeur D, Cesselin B, Liebl U, Vos MH, Fernandez A, Brun C, et al. Discovery of intracellular heme-binding protein HrtR, which controls heme efflux by the conserved HrtB-HrtA transporter in Lactococcus lactis. J Biol Chem. 2012; 287:4752–4758.

[43] Zhou G, Yin J, Chen H, Hua Y, Sun L, Gao H. Combined effect of loss of the caa₃ oxidase and Crp regulation drives Shewanella to thrive in redox-stratified environments. ISME J. 2013; 7:1752–1763.

[44] Zhang Y, Guo K, Meng Q, Gao H. Nitrite modulates aminoglycoside tolerance by inhibiting cytochrome heme-copper oxidase in bacteria. Commun Biol. 2020; 3:269.

[45] Mi W, Li Y, Yoon SH, Ernst RK, Walz T, Liao M. Structural basis of MsbA-mediated lipopolysaccharide transport. Nature. 2017; 549:233–237.

[46] Lambert E, Mehdipour AR, Schmidt A, Hummer G, Perez C. Evidence for a trap-and-flip mechanism in a proton-dependent lipid transporter. Nat Commun. 2022; 13:1022.

[47] Benarroch JM, Asally M. The microbiologist’s guide to membrane potential dynamics. TIM. 2020; 28:304–314.

[48] Giri RP, Mukhopadhyay MK, Basak UK, Chakrabarti A, Sanyal MK, Runge B, et al. Continuous uptake or saturation—investigation of concentration and surface-packing-specific hemin interaction with lipid membranes. J Phys Chem B. 2018; 122:7547–7554.

[49] Shi M, Wan F, Mao Y, Gao H. Unraveling the mechanism for the viability deficiency of Shewanella oneidensis oxyR null mutant. J Bacteriol. 2015; 197:2179–2189.

[50] Jin M, Jiang Y, Sun L, Yin J, Fu H, Wu G, et al. Unique organizational and functional features of the cytochrome c maturation system in Shewanella oneidensis. PLoS One. 2013; 8:e75610.

[51] Meng Q, Liang H, Gao H. Roles of multiple KASIII homologues of Shewanella oneidensis in initiation of fatty acid synthesis and in cerulenin resistance. Biochim Biophys Acta. 2018; 1863:1153–1163.

[52] Gao H, Wang X, Yang ZK, Chen J, Liang Y, Chen H, et al. Physiological roles of ArcA, Crp, and EtrA and their interactive control on aerobic and anaerobic respiration in Shewanella oneidensis. PLoS One. 2010; 5:e15295.

[53] Wu L, Wang J, Tang P, Chen H, Gao H. Genetic and molecular characterization of flagellar assembly in Shewanella oneidensis. PLoS One. 2011; 6:e21479.

[54] Corts AD, Thomason LC, Gill RT, Gralnick JA. A new recombineering system for precise genome-editing in Shewanella oneidensis strain MR-1 using single-stranded oligonucleotides. Sci Rep. 2019; 9:39.

[55] Luo Q, Dong Y, Chen H, Gao H. Mislocalization of Rieske protein PetA predominantly accounts for the aerobic growth defect of tat mutants in Shewanella oneidensis. PLoS One. 2013; 8:e62064.

[56] Gao T, Shi M, Ju L, Gao H. Investigation into FlhFG reveals distinct features of FlhF in regulating flagellum polarity in Shewanella oneidensis. Mol Microbiol. 2015; 98:571–585.

[57] Glanville DG, Mullineaux-Sanders C, Corcoran CJ, Burger BT, Imam S, Donohue TJ, et al. A high-throughput method for identifying novel genes that influence metabolic pathways reveals new iron and heme regulation in Pseudomonas aeruginosa. mSystems. 2021; 6:00933–00920.

[58] Chen H, Luo Q, Yin J, Gao T, Gao H. Evidence for the requirement of CydX in function but not assembly of the cytochrome bd oxidase in Shewanella oneidensis. Biochim Biophys Acta. 2015; 1850:318–328.

[59] Sun W, Fan Y, Wan F, Tao YJ, Gao H. Functional irreplaceability of Escherichia coli and Shewanella oneidensis OxyRs is critically determined by intrinsic differences in oligomerization. mBio. 2022; 13:03497–03421.

[60] Nourooz-Zadeh J, Tajaddini-Sarmadi J, Wolff SP. Measurement of plasma hydroperoxide concentrations by the ferrous oxidation-xylenol Orange assay in conjunction with triphenylphosphine. Anal Biochem. 1994; 220:403–409.

[61] Gao T, Ju L, Yin J, Gao H. Positive regulation of the Shewanella oneidensis OmpS38, a major porin facilitating anaerobic respiration, by Crp and Fur. Sci Rep. 2015; 5:14263.

[62] Cook CE, Bergman MT, Cochrane G, Apweiler R, Birney E. The European Bioinformatics Institute in 2017: data coordination and integration. Nucleic Acids Res. 2017; 46: D21–D29.

[63] Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, et al. Highly accurate protein structure prediction with AlphaFold. Nature. 2021; 596:583–589.

[64] Jo S, Kim T, Im W. Automated builder and database of protein/membrane complexes for molecular dynamics simulations. PLoS One. 2007; 2:e880.

[65] Jo S, Kim T, Iyer VG, Im W. CHARMM-GUI: a web-based graphical user interface for CHARMM. J Comput Chem. 2008; 29:1859–1865.

[66] Páll S, Zhmurov A, Bauer P, Abraham M, Lundborg M, Gray A, et al. Heterogeneous parallelization and acceleration of molecular dynamics simulations in GROMACS. J Chem Phys. 2020; 153:0018516.

[67] Trott O, Olson AJ. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem. 2010; 31:455–461.

[68] Bowers KJ, Chow DE, Xu H, Dror RO, Eastwood MP, Gregersen BA, et al. Scalable algorithms for molecular dynamics simulations on commodity clusters. in Proceedings of the 2006 ACM/IEEE conference on Supercomputing, 2006;84–es.

[69] Grant BJ, Rodrigues APC, ElSawy KM, McCammon JA, Caves LSD. Bio3d: an R package for the comparative analysis of protein structures. Bioinformatics. 2006; 22:2695–2696.

[70] Humphrey W, Dalke A, Schulten K. VMD: visual molecular dynamics. J Mol Graphics. 1996; 14:33–38.

[71] Schrodinger LLC. The PyMOL Molecular Graphics System, Version 1.8. 2023.

RIGHTS & PERMISSIONS

2025 The Author(s). mLife published by John Wiley & Sons Australia, Ltd on behalf of Institute of Microbiology, Chinese Academy of Sciences.