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

Abstract

In nature, methane is oxidized to methanol by two enzymes, the iron-dependent soluble methane monooxygenase (sMMO) and the copper-dependent particulate MMO (pMMO). While sMMO's diiron metal active site is spectroscopically and structurally well-characterized, pMMO's copper sites are not. Recent EPR and ENDOR studies have established the presence of two monocopper sites, but the coordination environment of only one has been determined, that within the PmoB subunit and denoted Cu B . Moreover, this recent work only focused on a type I methanotrophic pMMO, while previous observations of the type II enzyme were interpreted in terms of the presence of a dicopper site. First, this report shows that the type II Methylocystis species strain Rockwell pMMO, like the type I pMMOs, contains two monocopper sites and that its Cu B site has a coordination environment identical to that of type I enzymes. As such, for the full range of pMMOs this report completes the refutation of prior and ongoing suggestions of multicopper sites. Second, and of primary importance, EPR/ENDOR measurements (a) for the first time establish the coordination environment of the spectroscopically observed site, provisionally denoted Cu C , in both types of pMMO, thereby (b) establishing the assignment of this site observed by EPR to the crystallographically observed metal-binding site in the PmoC subunit. Finally, these results further indicate that Cu C is the likely site of biological methane oxidation by pMMO, a conclusion that will serve as a foundation for proposals regarding the mechanism of this reaction.

Recovery of particulate methane monooxygenase structure and activity in a lipid bilayer

“…The Cu B site is unequivocally mononuclear (fig. S8), in contrast to a recent cryo-EM structure of M. capsulatus (Bath) pMMO in DDM ( 26) but consistent with recent spectroscopic (23,24,27) and computational (25) studies. The corresponding Cu B sites in the cryo-EM structures of pMMO from Methylocystis sp.…”

“…Copper coordination by the side-chain oxygen of asparagine or glutamine is unusual but not unprecedented (33)(34)(35)(36). Recent X-band electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopic studies of M. capsulatus (Bath) pMMO in POPC nanodiscs indicate the presence of two histidine ligands to the copper ion spectroscopically assigned as Cu C (27), a finding that would be consistent with either the structural Cu C or Cu D site.…”

A Copper Cage‐Complex as Mimic of the pMMO CuC Site

“…As there are differing protocols for preparatory treatment, researchers draw contradictory conclusions on the structure and location of the active site, and accordingly also on the operating principle for methane hydroxylation. Even within the last year several opposing reports have been published on that topic [6–8] …”

“…Even within the last year several opposing reports have been published on that topic. [6][7][8] Copper occupancy of PmoC was shown to be crucial for catalytic activity in 2019 already, [9] and very recently again by cryo-electron microscopy studies on samples that still showed catalytic activity. [8] The activity goes along with an unusually high preservation of the enzyme structure, which also enables observation of the PmoC region by imaging techniques, and that for the first time in the absence of zinc.…”

Ein Kupfer‐Käfig‐Komplex als Nachahmung der pMMO CuC‐Seite

“…Rockwell pMMO (440 MHz) überein, welcher der Cu C ‐Seite zugeordnet wird. Auch die g‐Werte, die wir nach Zugabe von Wasser erhalten (2.05, 2.08 und 2.31), zeigen eine sehr hohe Übereinstimmung mit den berichteten Werten für Cu C (2.05, 2.07 und 2.31, [6] für einen Vergleich siehe Abbildung 5 unten). Dieses Verhalten lässt sich sehr gut durch die Beobachtungen früherer kristallographischer Analysen und 1 H‐ENDOR‐Spektroskopiestudien erklären, die ein Wassermolekül in der zweiten Koordinationssphäre von Cu C ausweisen, was eine ähnlich schwache Wechselwirkung von [1‐Cu]PF 6 mit Wasser aufzeigt und damit wieder eine große Gemeinsamkeit bedeutet.…”