The 5,10-methenyltetrahydromethanopterin hydrogenase (or Hmd), the so-called iron-sulfur cluster-free hydrogenase, is an

enzyme Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products ...

found in methanogenic

archea Archaea ( ; singular archaeon ) is a domain of single-celled organisms. These microorganisms lack cell nuclei and are therefore prokaryotes. Archaea were initially classified as bacteria, receiving the name archaebacteria (in the Archaeb ...

such as ''

Methanothermobacter marburgensis Methanothermobacter marburgensis is a thermophilic and obligately autotrophic archaeon. Its type strain is MarburgT. Its genome has been sequenced. It is named after the city of Marburg in Germany, where it was isolated from sewage sludge, but ...

''. It was discovered and first characterized by the Thauer group at the

Max Planck Institute Max or MAX may refer to: Animals * Max (dog) (1983–2013), at one time purported to be the world's oldest living dog * Max (English Springer Spaniel), the first pet dog to win the PDSA Order of Merit (animal equivalent of OBE) * Max (gorilla) ...

in Marburg. Hydrogenases are enzymes that either reduce protons or oxidize molecular dihydrogen.

Enzyme function

Methanogens rely on such enzymes to

catalyze Catalysis () is the process of increasing the rate of a chemical reaction by adding a substance known as a catalyst (). Catalysts are not consumed in the reaction and remain unchanged after it. If the reaction is rapid and the catalyst recyc ...

the reduction of CO₂ to

methane Methane ( , ) is a chemical compound with the chemical formula (one carbon atom bonded to four hydrogen atoms). It is a group-14 hydride, the simplest alkane, and the main constituent of natural gas. The relative abundance of methane on Ea ...

. One step in methanogenesis entails conversion of a methenyl group (formic acid oxidation state) to a methylene group (formaldehyde oxidation state). Among the hydrogenase family of enzymes, Hmd is unique in that it does not directly reduce CO₂ to CH₄. The natural substrate of the enzyme is the organic compound methenyltetrahydromethanopterin. The organic compound includes a methenyl group bound to two tertiary

amide In organic chemistry, an amide, also known as an organic amide or a carboxamide, is a compound with the general formula , where R, R', and R″ represent organic groups or hydrogen atoms. The amide group is called a peptide bond when it i ...

s. The methenyl group originated as CO₂ before being incorporated into the substrate, which is catalytically reduced by H₂ to methylenetetrahydromethanopterin as shown. Eventually the methylene group is further reduced and released as a molecule of methane. The

hydride In chemistry, a hydride is formally the anion of hydrogen( H−). The term is applied loosely. At one extreme, all compounds containing covalently bound H atoms are called hydrides: water (H2O) is a hydride of oxygen, ammonia is a hydride ...

transfer has also been shown to be stereospecific. Given that the substrate is planar the hydride originating from H₂ is always added to the pro-R face. In the reverse reaction stereospecificity is maintained and the highlighted hydride is removed.

Chemical and physical properties

Hmd holoenzyme

The Hmd holoenzyme includes the protein

homodimer In biochemistry, a protein dimer is a macromolecular complex formed by two protein monomers, or single proteins, which are usually non-covalently bound. Many macromolecules, such as proteins or nucleic acids, form dimers. The word ''dimer'' has ...

as well as its associated iron-containing co-factor. Several species of methanogens have been characterized that express enzymes in the Hmd hydrogenase family. Between species the enzyme is found with differing numbers of sub-units and some minor amino acid sequence variations. The monomer is approximately 45,000 Da in mass, although this value varies from species to species. The enzymatic activity of the enzyme is lost upon exposure to sunlight or UV. Photolysis causes the release of an iron atom and two molecules of carbon monoxide. In the holoenzyme the Fe and CO molecules are found associated with a 542 Da cofactor.

Hmd iron-containing cofactor

The iron-containing co-factor is found tightly associated with the protein. It can be released upon denaturation with

2-mercaptoethanol 2-Mercaptoethanol (also β-mercaptoethanol, BME, 2BME, 2-ME or β-met) is the chemical compound with the formula HOCH2CH2SH. ME or βME, as it is commonly abbreviated, is used to reduce disulfide bonds and can act as a biological antioxidant by s ...

or guanidine hydrochloride. Expression of the Hmd gene in '' E. coli'' without the co-factor results in an inactive holoenzyme. However, hydrogenase activity can be rescued by the addition of the iron-containing co-factor taken from denatured active enzyme. As mentioned, irradiation of the co-factor with UV light results in the loss of CO and Fe. In addition the 542 Da compound can be further degraded by a

phosphodiesterase A phosphodiesterase (PDE) is an enzyme that breaks a phosphodiester bond. Usually, ''phosphodiesterase'' refers to cyclic nucleotide phosphodiesterases, which have great clinical significance and are described below. However, there are many ot ...

(which specifically cleaves phosphate bonds). Hydrolysis of the phosphate bonds generates the

ribonucleotide In biochemistry, a ribonucleotide is a nucleotide containing ribose as its pentose component. It is considered a molecular precursor of nucleic acids. Nucleotides are the basic building blocks of DNA and RNA. Ribonucleotides themselves are basic ...

guanosine monophosphate and a modified 2-pyridone. On the basis of spectroscopic characterization, Shima et al. have proposed a structure for this organic cofactor (minus the iron atom and CO molecules) as shown: Although the mechanism by which Hmd acts is unknown, the iron-containing cofactor is in part responsible for the catalytic activity. High concentrations of CO inhibit the enzyme as well, implicating iron as the center of catalysis. It has been proposed that the iron functions to bind H₂ and the substrate methenyltetrahydromethanopterin, organizing these two reactants in close proximity.

Enzyme function

Chemical and physical properties

Hmd holoenzyme

Hmd iron-containing cofactor

References

Further reading