Oxidation of methane by a biological dicopper centre

Abstract

Vast world reserves of methane gas are underutilized as a feedstock for production of liquid fuels and chemicals due to the lack of economical and sustainable strategies for selective oxidation to methanol1. Current processes to activate the strong C–H bond (104 kcal/mol) in methane require high temperatures, are costly and inefficient, and produce waste2. In nature, methanotrophic bacteria perform this reaction under ambient conditions using metalloenzymes called methane monooxygenases (MMOs). MMOs are thus the optimal inspiration for an efficient, green catalyst3. There are two types of MMOs. Soluble MMO (sMMO), which is expressed by several strains of methanotrophs under copper limited conditions, oxidizes methane with a well characterized catalytic diiron center4. Particulate methane monooxygenase (pMMO), an integral membrane metalloenzyme produced by all methanotrophs, is composed of three subunits, pmoA, pmoB, and pmoC, arranged in a trimeric α3β3γ3 complex5. Despite 20 years of research and the availability of two crystal structures, the metal composition and location of the pMMO metal active site are not known. Here we show that pMMO activity is dependent on copper, not iron, and that the copper active site is located in the soluble domains of the pmoB subunit rather than within the membrane. Recombinant soluble fragments of pmoB (spmoB) bind copper and exhibit propylene and methane oxidation activities. Disruption of each copper center in spmoB by mutagenesis indicates that the active site is a dicopper center. These findings resolve the pMMO controversy and provide a promising new approach to developing environmentally friendly C–H oxidation catalysts.

Inactivation of the particulate methane monooxygenase (pMMO) in Methylococcus capsulatus (Bath) by acetylene

“…In the latter work [8], the labeling experiment is not clean, leading to the observation of [ 14 C]-radioactivity in the protein band corresponding to the PmoB subunit as well as other cytoplasmic proteins ( Figure 3 of ref 18) Subsequently, the "~27 kDa" subunit was assigned to PmoA by N-terminal sequencing [8]. Accordingly, there has been general consensus that the active site of the enzyme is associated with the PmoA subunit (not PmoB as mentioned in [14]). In the present study, however, we find only evidence for chemical modification of the PmoC subunit by ketene in the MALDI-TOF MS and LC-MS/MS experiments.…”

“…In light of these conflicting results, the location of the catalytic site and the mechanism of methane oxidation in pMMO are still under debate [1,14,17].…”

“…On the basis of the crystal structures available, it has been suggested that the active site of the enzyme resides at the water-exposed dicopper site of PmoB (Scheme 1) [5,14]. However, based on other biophysical and biochemical studies, the site of hydrocarbon hydroxylation has been implicated to be at site D within the transmembrane domain of the X-ray structure (Scheme 1) [1,15,16].…”

Mutagenesis of a Copper P-Type ATPase Encoding Genein Methylococcus capsulatus (Bath) Results inCopper-Resistance

“…sMMO is expressed at low copper-to-biomass ratios and pMMO is expressed at high copper to-biomass ratios growth conditions [9]. Copper also increases the synthesis of an extensive network of intracytoplasmic membranes [10] and is the active center metal of pMMO [11] Little is known about copper homeostasis in M. capsulatus although copper has a significant physiological role in this methanotroph. The genome sequencing of M. capsulatus led to the identification of four copper transport homologues [12].…”

Physiological Features during the Copper Switch in Methylococcus Capsulatus (Bath)

“…pMMO is expressed at high copper to-biomass ratios whereas sMMO is expressed at low copper-to-biomass ratios growth conditions [8]. Furthermore, copper enhances the synthesis of an extensive network of intracytoplasmic membranes [11] and is the active center metal of pMMO [12].…”