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The di-heme enzyme MauG catalyzes the biosynthesis of tryptophan tryptophylquinone (TTQ), the protein-derived cofactor of methylamine dehydrogenase (MADH). This process requires the sixelectron oxidation of a 119 kDa MADH precursor protein with incompletely synthesized TTQ (PreMADH). The kinetic mechanism of the initial two-electron oxidation of this natural substrate by MauG was characterized. The relative reactivity of free MauG towards H 2 O 2 and the O 2 analog CO was essentially the same as that of MauG in the pre-formed enzyme-substrate complex. The addition of H 2 O 2 to di-ferric MauG generated a di-heme bis-Fe(IV) species (i.e., Fe(IV)=O/Fe(IV)) which formed at a rate >300 s -1 and spontaneously returned to the di-ferric state at a rate of 2×10 -4 s -1 in the absence of substrate. The reaction of bis-Fe(IV) MauG with PreMADH exhibited saturation behavior with a limiting first-order rate constant of 0.8 s -1 and a K d ≤ 1.5 μM for the MauG-PreMADH complex. The results were the same whether bis-Fe(IV) MauG was mixed with PreMADH, or H 2 O 2 was added to the pre-formed enzyme-substrate complex to generate bis-Fe(IV) MauG followed by reaction with PreMADH. Stopped-flow kinetic studies of the reaction of di-ferrous MauG with CO yielded a faster major transition with a bimolecular rate constant of 5.4 × 10 5 M -1 s -1 , and slower transition with a rate 16 s -1 which was independent of [CO]. The same rates were obtained for binding of CO to di-ferrous MauG in complex with PreMADH. This demonstration of a random kinetic mechanism for the first two-electron oxidation reaction of MauG-dependent TTQ biosynthesis, in which the order of addition of oxidizing equivalent and substrate does not matter, is atypical of those of heme-dependent oxygenases that are not generally reactive towards oxygen in the absence of substrate. This kinetic mechanism is also distinct from that of the homologous di-heme cytochrome c peroxidases that require a mixed valence state for activity.Tryptophan tryptophylquinone (TTQ) 1 (1) is the protein-derived cofactor (2) of methylamine dehydrogenase (MADH), a 119 kDa heterotetrameric α 2 β 2 protein with a TTQ present on each β subunit (3,4). TTQ biosynthesis requires incorporation of two oxygens into βTrp57 and crosslinking of the indole rings of βTrp57 and βTrp108. This is not a self-processing event but requires the action of at least one processing enzyme. Deletion of mauG, a gene in the methylamine utilization (mau) gene cluster (5), allowed isolation of a biosynthetic precursor of MADH (PreMADH) in which βTrp57 is mono-hydroxylated at C7 and the cross-link is absent (6,7). MauG is a 42.3 kDa enzyme containing two c-type hemes (8). It exhibits homology to di-heme cytochrome c peroxidase (CCP) (9,10) but exhibits significant differences in catalytic and redox behavior (11,12). MauG catalyzes the six-electron oxidation of PreMADH to yield oxidized MADH with the mature protein-derived TTQ cofactor (Scheme 1). MauG- Given the complexity of the overall reaction of MauG-dependent TTQ ...

MauG is a diheme protein that catalyzes the six-electron oxidation of a biosynthetic precursor protein of methylamine dehydrogenase (PreMADH) with partially synthesized tryptophan tryptophylquinone (TTQ) to yield the mature protein with the functional protein-derived TTQ cofactor. The biosynthetic reaction proceeds via a relatively stable high valent bis-Fe(IV) intermediate. Oxidizing equivalents ([O]) for this reaction may be provided by either O 2 plus electrons from an external donor, or H 2 O 2 . The presence or absence of PreMADH has no influence on the reactivity of MauG with [O], however it is demonstrated that MauG is inactivated when supplied with [O] in the absence of PreMADH. The mechanism of inactivation appears to differ depending on the source of [O]. Repeated reaction of diferrous MauG with O 2 leads to loss of activity but not inactivation of heme, as judged by absorption spectroscopy and pyridine hemochrome assay. Repeated reaction of diferric MauG with H 2 O 2 leads to loss of activity and inactivation of heme, as well as some covalent cross-linking of MauG molecules. None of these deleterious effects with either source of [O] are observed when PreMADH is present to react with MauG. The radical scavenger hydroxyurea, and small molecule mimics of the monohydroxylated Trp residue of PreMADH also reacted with bis-Fe(IV) MauG and afforded protection against inactivation. These results demonstrate that while O 2 and H 2 O 2 readily react with MauG in the absence of PreMADH, the presence of this substrate is necessary to prevent suicide inactivation of MauG after formation of the bis-Fe(IV) intermediate.MauG is a 42.3 kDa enzyme containing two c-type hemes (1). It exhibits homology to diheme cytochrome c peroxidase (CCP) (2,3), but displays significant differences in catalytic and redox behavior (4,5). MauG catalyzes the final steps in the biosynthesis of tryptophan tryptophylquinone (TTQ) (6), the protein-derived cofactor (7) of methylamine dehydrogenase (MADH). MADH exhibits a 119 kDa heterotetrameric α 2 β 2 structure (8) and contained in each β subunit is a TTQ. TTQ is synthesized through posttranslational modification of two endogenous tryptophan residues of MADH. This modification involves crosslinking of βTrp57 and βTrp108, and the incorporation of two oxygens into βTrp57. It has been shown that the incorporation of the second oxygen into βTrp57 and the crosslinking reaction are catalyzed by MauG (1) (Scheme 1). Deletion of mauG, a gene in the methylamine utilization (mau) gene cluster (9), allowed isolation of a biosynthetic precursor protein of MADH (PreMADH) in which βTrp57 is monohydroxylated at C7 and the cross-link is absent (10,11 Experimental ProceduresThe methods for homologous expression of MauG in Paracoccus denitrificans and its purification were as described previously (1). PreMADH, the biosynthetic precursor of MADH with incompletely synthesized TTQ, which contains monohydroxylated βTrp57 and no crosslink to βTrp108 (11), was heterologously expressed in Rhodobacter sph...

The redox regulation of Janus kinase 2 (JAK2) is poorly understood, and there are contradictory reports as to whether the enzyme's activity is inhibited or stimulated by oxidizing conditions in the cell. Here we demonstrate that multiple cysteine residues within the JAK2 catalytic domain may be crucial for enzymatic activity. The enzyme is catalytically inactive when oxidized; activity can be restored via reduction to the thiol state. A series of recombinant variants of JAK2 were overproduced using the baculoviral expression vector system. A truncated variant of JAK2, GST/(NDelta661)rJAK2, provided evidence that the amino-terminal autoinhibitory domain was not essential for direct redox regulation and that only nine cysteine residues were potentially involved. The effect of individually and combinatorially altering these nine cysteines was examined via cysteine-to-serine mutagenesis. This identified four cysteine residues in the catalytic domain (Cys866, Cys917, Cys1094, and Cys1105) that cooperatively maintain JAK2's catalytic competency. Our data are consistent with a direct mechanism for redox regulation of JAK2 via oxidation and reduction of critical cysteine residues.