5,10-methenyltetrahydromethanopterin hydrogenase
Encyclopedia
The 5,10-methenyltetrahydromethanopterin hydrogenase (or Hmd), the so-called iron-sulfur cluster-free hydrogenase
, is an enzyme
found in methanogenic archea such as Methanothermacter marburgensis. It was discovered and first characterized by the Thauer group at the Max Planck Institute in Marburg. Hydrogenases are enzymes that either reduce protons or oxidize molecular dihydrogen.
. 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
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
transfer has also been shown to be stereospecific. Given that the substrate is planer 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.
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.
with 2-mercaptoethanol
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
(which specifically cleaves phosphate bonds). Hydrolysis of the phosphate bonds generates the ribonucleotide
guanine mono-phosphate 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.
Hydrogenase
A hydrogenase is an enzyme that catalyses the reversible oxidation of molecular hydrogen . Hydrogenases play a vital role in anaerobic metabolism....
, is an enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
found in methanogenic archea such as Methanothermacter marburgensis. It was discovered and first characterized by the Thauer group at the Max Planck Institute in Marburg. Hydrogenases are enzymes that either reduce protons or oxidize molecular dihydrogen.
Enzyme function
Methanogens rely on such enzymes to catalyze the reduction of CO2, to methaneMethane
Methane is a chemical compound with the chemical formula . It is the simplest alkane, the principal component of natural gas, and probably the most abundant organic compound on earth. The relative abundance of methane makes it an attractive fuel...
. 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
Amide
In chemistry, an amide is an organic compound that contains the functional group consisting of a carbonyl group linked to a nitrogen atom . The term refers both to a class of compounds and a functional group within those compounds. The term amide also refers to deprotonated form of ammonia or an...
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.
Hydride
In chemistry, a hydride is the anion of hydrogen, H−, or, more commonly, a compound in which one or more hydrogen centres have nucleophilic, reducing, or basic properties. In compounds that are regarded as hydrides, hydrogen is bonded to a more electropositive element or group...
transfer has also been shown to be stereospecific. Given that the substrate is planer 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.
Hmd Holoenzyme
The Hmd holoenzyme includes the protein homodimer 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 co-factor
The iron-containing co-factor is found tightly associated with the protein. It can be released upon denaturationDenaturation (biochemistry)
Denaturation is a process in which proteins or nucleic acids lose their tertiary structure and secondary structure by application of some external stress or compound, such as a strong acid or base, a concentrated inorganic salt, an organic solvent , or heat...
with 2-mercaptoethanol
2-Mercaptoethanol
2-Mercaptoethanol is the chemical compound with the formula HOCH2CH2SH. It is a hybrid of ethylene glycol, HOCH2CH2OH, and 1,2-ethanedithiol, HSCH2CH2SH...
or guanidine hydrochloride. Expression of the Hmd gene in E. coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
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
Phosphodiesterase
A phosphodiesterase is any enzyme that breaks a phosphodiester bond. Usually, people speaking of phosphodiesterase are referring to cyclic nucleotide phosphodiesterases, which have great clinical significance and are described below...
(which specifically cleaves phosphate bonds). Hydrolysis of the phosphate bonds generates the ribonucleotide
Ribonucleotide
A ribonucleotide or ribotide is a nucleotide in which a purine or pyrimidine base is linked to a ribose molecule and exactly one phosphate group. In living organisms the most common bases for ribonucleotides are adenine , guanine , cytosine , or uracil ....
guanine mono-phosphate and a modified 2-pyridone
2-Pyridone
2-Pyridone is an organic compound with the formula . This colourless crystalline solid is used in peptide synthesis. It is well known to form hydrogen bonded structures somewhat related to the base-pairing mechanism found in RNA and DNA...
. 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: