Capturing a bis-Fe(IV) State in Methylosinus trichosporium OB3b MbnH

Abstract

The diheme bacterial cytochrome c peroxidase (bCcP)/MauG superfamily is a diverse set of enzymes that remains largely uncharacterized. One recently discovered member, MbnH, converts a tryptophan residue in its substrate protein, MbnP, to kynurenine. Here we show that upon reaction with H 2 O 2 , MbnH forms a bis-Fe(IV) intermediate, a state previously detected in just two other enzymes, MauG and BthA. Using absorption, Mossbauer, and electron paramagnetic resonance (EPR) spectroscopies coupled with kinetic analysis, we characterized the bis-Fe(IV) state of MbnH and determined that this intermediate decays back to the diferric state in the absence of MbnP substrate. In the absence of MbnP substrate, MbnH can also detoxify H 2 O 2 to prevent oxidative self damage, unlike MauG, which has long been viewed as the prototype for bis-Fe(IV) forming enzymes. MbnH performs a different reaction from MauG, while the role of BthA remains unclear. All three enzymes can form a bis-Fe(IV) intermediate but within distinct kinetic regimes. The study of MbnH significantly expands our knowledge of enzymes that form this species. Computational and structural analyses indicate that electron transfer between the two heme groups in MbnH and between MbnH and the target tryptophan in MbnP likely occurs via a hole-hopping mechanism involving intervening tryptophan residues. These findings set the stage for discovery of additional functional and mechanistic diversity within the bCcP/MauG superfamily.

Direct Methane Oxidation by Copper- and Iron-Dependent Methane Monooxygenases

“… , Due to its high affinity for Cu­(I), Mbn not only binds copper in solution but can also extract copper from mineral sources or glass. ,, The mechanism of Mbn secretion has not been established but is proposed to involve MbnM, a member of the multidrug and toxic compound extrusion family. , Uptake of copper-loaded Mbn is an active transport process mediated by the outer membrane TonB-dependent transporter MbnT, which is encoded both within Mbn operons and elsewhere in the methanotroph genomes. − After intact copper-loaded Mbn enters the cell, it may interact with periplasmic proteins such as MbnE or MbnP, followed by import to the cytoplasm, perhaps by ABC transporters (Figure ). MbnP was recently shown to bind Cu­(I) using a kynurenine residue that is generated by the diheme enzyme MbnH. − The MbnP and MbnH genes are typically found adjacent to genes encoding MbnT. It is not known how copper is then delivered to pMMO or cytoplasmic cellular targets, including transcription factors.…”