Escherichia coli K-12 substr. MG1655 Polypeptide: hydrogenase 2 - integral membrane subunit HybB

Gene: hybB Accession Numbers: EG11800 (EcoCyc), b2995, ECK2989

Regulation Summary Diagram

Regulation summary diagram for hybB

Component of: hydrogenase 2 (extended summary available)

The HybB protein is predicted to be an integral membrane component of hydrogenase 2 [Menon94]. hybB contains a HXXH conserved motif associated with cytochrome b type proteins [Menon94] HybB contains no conserved hisitidines that would serve as heme iron ligands [Dubini02]. HybB may act as a proton pump during H(2):quinone oxidoreductase activity [Pinske15]

A hybB in-frame deletion mutant can not grow on glycerol and fumarate as the sole energy sources. However, the HybOHybC complex is correctly targeted to the membrane and active with the artificial electron acceptor benzyl viologen (BV) [Dubini02].

Gene Citations: [Sargent98]

Locations: inner membrane

Map Position: [3,141,008 <- 3,142,186] (67.7 centisomes, 244°)
Length: 1179 bp / 392 aa

Molecular Weight of Polypeptide: 43.602 kD (from nucleotide sequence)

Unification Links: ASAP:ABE-0009830, CGSC:33414, EchoBASE:EB1748, EcoGene:EG11800, EcoliWiki:b2995, OU-Microarray:b2995, PortEco:hybB, Protein Model Portal:P37180, RefSeq:NP_417469, RegulonDB:EG11800, String:511145.b2995, UniProt:P37180

Relationship Links: InterPro:IN-FAMILY:IPR005614, Pfam:IN-FAMILY:PF03916

In Paralogous Gene Group: 245 (2 members)

Gene-Reaction Schematic

Gene-Reaction Schematic

Genetic Regulation Schematic

Genetic regulation schematic for hybB

GO Terms:
Biological Process:
Inferred from experimentGO:0009061 - anaerobic respiration [Menon94, Dubini02]
Inferred by computational analysisGO:0055114 - oxidation-reduction process [UniProtGOA11a]
Molecular Function:
Inferred from experimentGO:0033748 - hydrogenase (acceptor) activity [Dubini02]
Inferred by computational analysisGO:0046872 - metal ion binding [UniProtGOA11a]
Cellular Component:
Inferred from experimentInferred by computational analysisGO:0005886 - plasma membrane [UniProtGOA11, UniProtGOA11a, DiazMejia09, Daley05]
Inferred from experimentGO:0044569 - [Ni-Fe] hydrogenase complex [Dubini02]
Inferred by computational analysisGO:0005887 - integral component of plasma membrane [Menon94]
Inferred by computational analysisGO:0016020 - membrane [UniProtGOA11a]
Inferred by computational analysisGO:0016021 - integral component of membrane [UniProtGOA11a, Gaudet10]

MultiFun Terms: cell structuremembrane
metabolismbiosynthesis of macromolecules (cellular constituents)large molecule carrierscytochromes
metabolismenergy metabolism, carbonanaerobic respiration
metabolismenergy production/transportelectron donors

Essentiality data for hybB knockouts:

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB enrichedYes 37 Aerobic 6.95   Yes [Gerdes03, Comment 1]
LB LennoxYes 37 Aerobic 7   Yes [Baba06, Comment 2]
M9 medium with 1% glycerolYes 37 Aerobic 7.2 0.35 Yes [Joyce06, Comment 3]
MOPS medium with 0.4% glucoseYes 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 2]

Last-Curated 15-Jan-2015 by Mackie A, Macquarie University

Subunit of: hydrogenase 2

Synonyms: HYD2, hydrogenase-2, hydrogen:menaquinone oxidoreductase 2

Subunit composition of hydrogenase 2 = [HybA][HybB][HybO][HybC]
         hydrogenase 2 - [Fe-S] binding, ferredoxin-type component HybA = HybA (summary available)
         hydrogenase 2 - integral membrane subunit HybB = HybB (summary available)
         hydrogenase 2, small subunit = HybO (summary available)
         hydrogenase 2, large subunit = HybC (summary available)

Hydrogenase 2 is a membrane-bound, [Ni-Fe] enzyme produced under anaerobic conditions. Hydrogenase 2 is a respiratory enzyme which couples hydrogen oxidation in the periplasm to reduction of the inner membrane quinone pool [Ballantine86, Sargent98]. Hydrogenase 2 participates in H(2) dependent reduction of fumarate, dimethyl sulfoxide and trimethylamine N-oxide [Sawers85, Laurinavichene01, Pinske15] (and see [Unden97].

Hydrogenase 2 is an oxygen sensitive enzyme - it is unable to catalyse H(2) oxidation under aerobic conditions [Laurinavichene01, Lukey10]. Hydrogenase 2 functions optimally at redox potentials lower than -100 to -150 mV [Laurinavichene02, Lukey10]. Hydrogenase 2 is capable of bidirectional catalysis in vitro [Lukey10] and in vivo [Pinske15]. Hydrogenase 2 can function as an H(2) evolving enzyme (ie. as a proton reductant) during fermentative growth with glycerol; this endergonic reaction is driven by the membrane proton gradient and probably functions to prevent over reduction of the quinone pool [Pinske15].

Hydrogenase 2 uses menaquinone/demethylmenaquinone to couple hydrogen oxidation to fumarate reduction during anaerobic respiratory growth with glycerol and fumarate and also during H(2) evolution during fermentation with glycerol; hydrogenase 2 can rapidly switch between H(2) evolution and H(2) oxidation modes in vivo [Pinske15].

Trypsin treatment of membranes releases an active, soluble fragment of hydrogenase 2 which consists of the large and small subunits [Ballantine86]. Hydrogenase 2 is encoded within the hyb operon (hybGFEDCBAO); the complete enzyme complex is thought to consist of the HybA, HybB, HybC and HybO subunits [Menon94, Dubini02]. HybOC forms the core catalytic dimer anchored to the membrane via a hydrophobic helix at the C-terminus of HybO; HybA (a ferredoxin type protein) and HybB (an integral membrane protein) are essential for shuttling electrons to the quinone pool [Dubini02, Pinske15].

HybC and HybO are coordinately assembled and processed; acquisition of the [NiFe] cofactor, C-terminal processing of HybC and subsequent association with the small subunit (HybO) are required prior to export by the Tat system [Rodrigue96, Sargent98, Rodrigue99, Zhang03d, Dubini03]. Maturation and membrane targeting of hydrogenase 2 involves proteins encoded within the hyp and hyb operons ( HypB, HypD, HypE, HybD, HybE and HybG) and the HypF protein (reviews: [Bock06, Forzi07]).

Expression of the hyb operon is induced under anaerobic conditions and repressed by nitrate [Richard99].

E. coli K-12 contains a second membrane associated hydrogenase - hydrogenase 1 - and a third hydrogenase - hydrogenase 3 - which is part of the formate hydrogenlyase complex. A potential fourth hydrogenase - hydrogenase 4 - is encoded within the hyf operon.

Reviews: [Sawers94, Vignais04]

Citations: [Pinske11, Ballantine85 , Trchounian14]

Locations: periplasmic space, inner membrane

GO Terms:
Biological Process:
Inferred from experimentGO:0009061 - anaerobic respiration [Pinske15, Menon94, Sawers85]
Inferred from experimentGO:0019588 - anaerobic glycerol catabolic process [Pinske15]
Inferred from experimentGO:0019645 - anaerobic electron transport chain [Laurinavichene01, Pinske15]
Inferred from experimentGO:1902421 - hydrogen metabolic process [Sawers85]
Molecular Function:
Inferred from experimentGO:0005506 - iron ion binding [Ballantine86]
Inferred from experimentGO:0009055 - electron carrier activity [Laurinavichene01]
Inferred from experimentGO:0016151 - nickel cation binding [Ballantine86]
Inferred from experimentGO:0033748 - hydrogenase (acceptor) activity [Laurinavichene02, Sargent98, Ballantine86]
Inferred from experimentGO:0047067 - hydrogen:quinone oxidoreductase activity [Pinske15]
Cellular Component:
Inferred from experimentGO:0031236 - extrinsic component of periplasmic side of plasma membrane [Rodrigue99]
Inferred by computational analysisInferred from experimentGO:0044569 - [Ni-Fe] hydrogenase complex [Dubini02, Menon94, Ballantine86]

Revised 12-Jan-2015 by Mackie A, Macquarie University
Last-Curated 07-Jan-2015 by Mackie A, Macquarie University

Enzymatic reaction of: hydrogen:menaquinone oxidoreductase (hydrogenase 2)

Inferred from experiment

EC Number:

Transport reaction diagram for hydrogen:menaquinone oxidoreductase

Alternative Substrates for a menaquinone: benzyl viologen [Ballantine86]

In Pathways: hydrogen to fumarate electron transfer, hydrogen to trimethylamine N-oxide electron transfer, hydrogen to dimethyl sulfoxide electron transfer

The representation of the hydrogenase 2 complex depicts the location of the donor (ie. H2) oxidation site and menaquinone reduction site at opposite sides of the membrane (H+/e- = 1). This representation has not been experimentally established.

Cofactors or Prosthetic Groups: a [FeS] iron-sulfur cluster [Sargent98], a nickel-iron-sulfur cluster [Ballantine86]

Inhibitors (Unknown Mechanism): Cu2+ [Ballantine86], N-bromosuccinimide [Ballantine86], Co2+ [Ballantine86]

Sequence Features

Protein sequence of hydrogenase 2 - integral membrane subunit HybB with features indicated

Feature Class Location Citations Comment
Transmembrane-Region 12 -> 32
Inferred by computational analysis[UniProt15]
UniProt: Helical.
Transmembrane-Region 35 -> 55
Inferred by computational analysis[UniProt15]
UniProt: Helical.
Transmembrane-Region 59 -> 79
Inferred by computational analysis[UniProt15]
UniProt: Helical.
Transmembrane-Region 91 -> 111
Inferred by computational analysis[UniProt15]
UniProt: Helical.
Transmembrane-Region 134 -> 154
Inferred by computational analysis[UniProt15]
UniProt: Helical.
Transmembrane-Region 169 -> 189
Inferred by computational analysis[UniProt15]
UniProt: Helical.
Transmembrane-Region 208 -> 228
Inferred by computational analysis[UniProt15]
UniProt: Helical.
Transmembrane-Region 250 -> 270
Inferred by computational analysis[UniProt15]
UniProt: Helical.
Transmembrane-Region 282 -> 302
Inferred by computational analysis[UniProt15]
UniProt: Helical.
Sequence-Conflict 306 -> 307
Inferred by curator[Menon94, UniProt15]
UniProt: (in Ref. 1; AAA21590/AAA69162).
Transmembrane-Region 334 -> 354
Inferred by computational analysis[UniProt15]
UniProt: Helical.
Transmembrane-Region 356 -> 376
Inferred by computational analysis[UniProt15]
UniProt: Helical.

Sequence Pfam Features

Protein sequence of hydrogenase 2 - integral membrane subunit HybB with features indicated

Feature Class Location Citations Comment
Pfam PF03916 45 -> 330
Inferred by computational analysis[Finn14]
NrfD : Polysulphide reductase, NrfD

Gene Local Context (not to scale -- see Genome Browser for correct scale)

Gene local context diagram

Transcription Unit

Transcription-unit diagram


10/20/97 Gene b2995 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG11800; confirmed by SwissProt match.


Baba06: Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006). "Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection." Mol Syst Biol 2;2006.0008. PMID: 16738554

Ballantine85: Ballantine SP, Boxer DH (1985). "Nickel-containing hydrogenase isoenzymes from anaerobically grown Escherichia coli K-12." J Bacteriol 163(2);454-9. PMID: 3894325

Ballantine86: Ballantine SP, Boxer DH (1986). "Isolation and characterisation of a soluble active fragment of hydrogenase isoenzyme 2 from the membranes of anaerobically grown Escherichia coli." Eur J Biochem 1986;156(2);277-84. PMID: 3516690

Bock06: Bock A, King PW, Blokesch M, Posewitz MC (2006). "Maturation of hydrogenases." Adv Microb Physiol 51;1-71. PMID: 17091562

Daley05: Daley DO, Rapp M, Granseth E, Melen K, Drew D, von Heijne G (2005). "Global topology analysis of the Escherichia coli inner membrane proteome." Science 308(5726);1321-3. PMID: 15919996

DiazMejia09: Diaz-Mejia JJ, Babu M, Emili A (2009). "Computational and experimental approaches to chart the Escherichia coli cell-envelope-associated proteome and interactome." FEMS Microbiol Rev 33(1);66-97. PMID: 19054114

Dubini02: Dubini A, Pye RL, Jack RL, Palmer T, Sargent F (2002). "How bacteria get energy from hydrogen: a genetic analysis of periplasmic hydrogen oxidationin Escherichia coli." Int J Hydrogen Energy 27(11-12);1413-1420.

Dubini03: Dubini A, Sargent F (2003). "Assembly of Tat-dependent [NiFe] hydrogenases: identification of precursor-binding accessory proteins." FEBS Lett 549(1-3);141-6. PMID: 12914940

Finn14: Finn RD, Bateman A, Clements J, Coggill P, Eberhardt RY, Eddy SR, Heger A, Hetherington K, Holm L, Mistry J, Sonnhammer EL, Tate J, Punta M (2014). "Pfam: the protein families database." Nucleic Acids Res 42(Database issue);D222-30. PMID: 24288371

Forzi07: Forzi L, Sawers RG (2007). "Maturation of [NiFe]-hydrogenases in Escherichia coli." Biometals 20(3-4):565-78. PMID: 17216401

Gaudet10: Gaudet P, Livstone M, Thomas P (2010). "Annotation inferences using phylogenetic trees." PMID: 19578431

Gerdes03: Gerdes SY, Scholle MD, Campbell JW, Balazsi G, Ravasz E, Daugherty MD, Somera AL, Kyrpides NC, Anderson I, Gelfand MS, Bhattacharya A, Kapatral V, D'Souza M, Baev MV, Grechkin Y, Mseeh F, Fonstein MY, Overbeek R, Barabasi AL, Oltvai ZN, Osterman AL (2003). "Experimental determination and system level analysis of essential genes in Escherichia coli MG1655." J Bacteriol 185(19);5673-84. PMID: 13129938

Joyce06: Joyce AR, Reed JL, White A, Edwards R, Osterman A, Baba T, Mori H, Lesely SA, Palsson BO, Agarwalla S (2006). "Experimental and computational assessment of conditionally essential genes in Escherichia coli." J Bacteriol 188(23);8259-71. PMID: 17012394

Laurinavichene01: Laurinavichene TV, Tsygankov AA (2001). "H2 consumption by Escherichia coli coupled via hydrogenase 1 or hydrogenase 2 to different terminal electron acceptors." FEMS Microbiol Lett 202(1);121-4. PMID: 11506918

Laurinavichene02: Laurinavichene TV, Zorin NA, Tsygankov AA (2002). "Effect of redox potential on activity of hydrogenase 1 and hydrogenase 2 in Escherichia coli." Arch Microbiol 178(6);437-42. PMID: 12420163

Lukey10: Lukey MJ, Parkin A, Roessler MM, Murphy BJ, Harmer J, Palmer T, Sargent F, Armstrong FA (2010). "How Escherichia coli is equipped to oxidize hydrogen under different redox conditions." J Biol Chem 285(6);3928-38. PMID: 19917611

Menon94: Menon NK, Chatelus CY, Dervartanian M, Wendt JC, Shanmugam KT, Peck HD, Przybyla AE (1994). "Cloning, sequencing, and mutational analysis of the hyb operon encoding Escherichia coli hydrogenase 2." J Bacteriol 176(14);4416-23. PMID: 8021226

Pinske11: Pinske C, Sawers G (2011). "Iron restriction induces preferential down-regulation of H2-consuming over H2-evolving reactions during fermentative growth of Escherichia coli." BMC Microbiol 11;196. PMID: 21880124

Pinske15: Pinske C, Jaroschinsky M, Linek S, Kelly CL, Sargent F, Sawers RG (2015). "Physiology and bioenergetics of [NiFe]-hydrogenase 2-catalyzed H2-consuming and H2-producing reactions in Escherichia coli." J Bacteriol 197(2);296-306. PMID: 25368299

Richard99: Richard DJ, Sawers G, Sargent F, McWalter L, Boxer DH (1999). "Transcriptional regulation in response to oxygen and nitrate of the operons encoding the [NiFe] hydrogenases 1 and 2 of Escherichia coli." Microbiology 145 ( Pt 10);2903-12. PMID: 10537212

Rodrigue96: Rodrigue A, Boxer DH, Mandrand-Berthelot MA, Wu LF (1996). "Requirement for nickel of the transmembrane translocation of NiFe-hydrogenase 2 in Escherichia coli." FEBS Lett 392(2);81-6. PMID: 8772179

Rodrigue99: Rodrigue A, Chanal A, Beck K, Muller M, Wu LF (1999). "Co-translocation of a periplasmic enzyme complex by a hitchhiker mechanism through the bacterial tat pathway." J Biol Chem 274(19);13223-8. PMID: 10224080

Sargent98: Sargent F, Ballantine SP, Rugman PA, Palmer T, Boxer DH (1998). "Reassignment of the gene encoding the Escherichia coli hydrogenase 2 small subunit--identification of a soluble precursor of the small subunit in a hypB mutant." Eur J Biochem 1998;255(3);746-54. PMID: 9738917

Sawers85: Sawers RG, Ballantine SP, Boxer DH (1985). "Differential expression of hydrogenase isoenzymes in Escherichia coli K-12: evidence for a third isoenzyme." J Bacteriol 164(3);1324-31. PMID: 3905769

Sawers94: Sawers G (1994). "The hydrogenases and formate dehydrogenases of Escherichia coli." Antonie Van Leeuwenhoek 1994;66(1-3);57-88. PMID: 7747941

Trchounian14: Trchounian A, Gary Sawers R (2014). "Novel insights into the bioenergetics of mixed-acid fermentation: can hydrogen and proton cycles combine to help maintain a proton motive force?." IUBMB Life 66(1);1-7. PMID: 24501007

Unden97: Unden G, Bongaerts J (1997). "Alternative respiratory pathways of Escherichia coli: energetics and transcriptional regulation in response to electron acceptors." Biochim Biophys Acta 1320(3);217-34. PMID: 9230919

UniProt15: UniProt Consortium (2015). "UniProt version 2015-08 released on 2015-07-22." Database.

UniProtGOA11: UniProt-GOA (2011). "Gene Ontology annotation based on the manual assignment of UniProtKB Subcellular Location terms in UniProtKB/Swiss-Prot entries."

UniProtGOA11a: UniProt-GOA (2011). "Gene Ontology annotation based on manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."

Vignais04: Vignais PM, Colbeau A (2004). "Molecular biology of microbial hydrogenases." Curr Issues Mol Biol 6(2);159-88. PMID: 15119826

Zhang03d: Zhang M, Pradel N, Mandrand-Berthelot MA, Yu Z, Wu LF (2003). "Effect of alteration of the C-terminal extension on the maturation and folding of the large subunit of the Escherichia coli hydrogenase-2." Biochimie 85(6);575-9. PMID: 12829374

Other References Related to Gene Regulation

Chung13: Chung D, Park D, Myers K, Grass J, Kiley P, Landick R, Keles S (2013). "dPeak: high resolution identification of transcription factor binding sites from PET and SET ChIP-Seq data." PLoS Comput Biol 9(10);e1003246. PMID: 24146601

MendozaVargas09: Mendoza-Vargas A, Olvera L, Olvera M, Grande R, Vega-Alvarado L, Taboada B, Jimenez-Jacinto V, Salgado H, Juarez K, Contreras-Moreira B, Huerta AM, Collado-Vides J, Morett E (2009). "Genome-wide identification of transcription start sites, promoters and transcription factor binding sites in E. coli." PLoS One 4(10);e7526. PMID: 19838305

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Please cite the following article in publications resulting from the use of EcoCyc: Nucleic Acids Research 41:D605-12 2013
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