Particulate Methane Monooxygenase and thePmoDProtein

Abstract

Particulate methane monooxygenase (pMMO) is the main enzyme used by methanotrophic bacteria to metabolize methane as their sole source of carbon and energy. pMMO is a 300‐kDa copper‐dependent, membrane‐bound metalloenzyme housed in specialized intracytoplasmic membrane (ICM) structures formed under copper‐replete growth conditions. Five crystal structures of pMMO from different methanotrophic species have revealed that pMMO comprises three subunits, PmoA, PmoB, and PmoC, arranged in a (αβγ) 3 cylindrical‐shaped trimer. Since the last article, it has been established that pMMO contains two monocopper sites, one each in the PmoB and PmoC subunits. Both sites are likely involved in methane oxidation, but their specific roles are yet to be fully determined. The PmoD protein is unique to methanotrophs and is often encoded within the pMMO operon. PmoD consists of a single transmembrane helix and a cupredoxin‐like periplasmic domain that has been crystallographically characterized. Although its function remains unknown, disruption of the gene encoding PmoD results in a growth defect under high copper conditions, suggesting a potential role in methane oxidation. Biochemical analysis indicates that the periplasmic domain of PmoD binds copper, forming an unusual Cu A site in vitro .

Coordination of the Copper Centers in Particulate Methane Monooxygenase: Comparison between Methanotrophs and Characterization of the CuCSite by EPR and ENDOR Spectroscopies

“…Given the saturated equatorial coordination found for Cu B and its presence as Cu­(II) even in vivo, it is not likely to be the site of dioxygen and methane binding. In support of this conclusion, the three Cu B histidine ligands are not conserved in a subset of pMMOs from type III methanotrophic verrucomicrobia …”

“…There is also a crystallographically identified metal-binding site in the PmoC subunit whose ligand set, which includes Asp129, His133, and His146 (Figure ), is strictly conserved in pMMOs from all types of methanotrophs . In some pMMO structures, these three ligands coordinate a zinc ion from the crystallization buffer. , In the absence of zinc, the site is occupied by copper. , Furthermore, this site is adjacent to a highly conserved region of PmoC that is disordered in all the crystal structures, and nTDMS fragmentation of PmoC was not successful, underscoring the dearth of information about the properties of this site .…”

“…capsulatus (Bath), a type I methanotroph, while the crystal structures guiding the interpretations include pMMOs from type II methanotrophs as well. These two methanotroph subclasses differ in cell morphologies, metabolic aspects, and lipid content, and their pMMOs exhibit structural differences, including an unidentified helix in the type II methanotroph pMMO structures . In addition, some type I methanotroph pMMOs contain a third “bis-His” copper site in the PmoB subunit that has not been observed in the type II methanotroph pMMOs. ,, …”

Towards a unified understanding of the copper sites in particulate methane monooxygenase: an X-ray absorption spectroscopic investigation

“…[1][2][3][4] Alternatively, methanotrophic bacteria 5 have evolved two genetically unrelated enzymes to catalyze methane to methanol conversion under ambient conditions: soluble and particulate methane monooxygenase (sMMO and pMMO, respectively). 6 sMMO features a well-characterized diiron active site, 7,8 and while pMMO is generally accepted as a copper enzyme, 6,9,10 the identity of its catalytic copper cofactor has proved far more elusive, severely impeding our mechanistic understanding of this important enzyme. 11 Much of the literature over the past two decades has focused on the possibility of a multinuclear copper active site, with the main proposals involving a tricopper site in the PmoA subunit and a dicopper site in the PmoB subunit (Fig.…”

“…9,[11][12][13] While a metal binding site in PmoA has never been observed crystallographically, crystal structures of pMMOs isolated from multiple methanotrophs all contain copper in PmoB (Cu B site), modeled as either dicopper or monocopper. 6 Dening the nuclearity and ligation of the pMMO copper cofactors is a fundamental step towards elucidating a catalytic mechanism.…”