Diradical intermediate within the context of tryptophan tryptophylquinone biosynthesis

Abstract

Despite the importance of tryptophan (Trp) radicals in biology, very few radicals have been trapped and characterized in a physiologically meaningful context. Here we demonstrate that the diheme enzyme MauG uses Trp radical chemistry to catalyze formation of a Trp-derived tryptophan tryptophylquinone cofactor on its substrate protein, premethylamine dehydrogenase. The unusual sixelectron oxidation that results in tryptophan tryptophylquinone formation occurs in three discrete two-electron catalytic steps. Here the exact order of these oxidation steps in the processive six-electron biosynthetic reaction is determined, and reaction intermediates are structurally characterized. The intermediates observed in crystal structures are also verified in solution using mass spectrometry. Furthermore, an unprecedented Trp-derived diradical species on premethylamine dehydrogenase, which is an intermediate in the first two-electron step, is characterized using high-frequency and -field electron paramagnetic resonance spectroscopy and UV-visible absorbance spectroscopy. This work defines a unique mechanism for radical-mediated catalysis of a protein substrate, and has broad implications in the areas of applied biocatalysis and understanding of oxidative protein modification during oxidative stress.cofactor biosynthesis | tryptophan radical | heme | posttranslational modification | electron transfer P rotein-based radicals, particularly on tyrosine (Tyr) and tryptophan (Trp) residues, have been implicated in a large number of catalytic and electron transfer reactions in biology (1), including the long-range electron transfer reactions required for photosynthesis (2), respiration (3), and DNA synthesis (4) and repair (5). Aberrant formation of protein radicals during oxidative stress is also of special importance. Evidence for the involvement of protein-based and substrate-based radicals in enzyme-catalyzed reactions has increased substantially in recent years, and expanded our knowledge of the scope of chemical reactions accessible to enzymes. The ability of enzymes to catalyze what were previously thought to be unattainable reactions is exemplified by MauG. The biosynthesis of the tryptophan tryptophylquinone (TTQ) cofactor in methylamine dehydrogenase (MADH) requires posttranslational modification of two tryptophan residues. MADH from Paracoccus denitrificans is a 119-kDa α 2 β 2 heterotetramer that contains two active sites and two TTQ cofactors derived from residues βTrp57 and βTrp108 (6). MauG is a c-type diheme enzyme that catalyzes the conversion of a MADH precursor (preMADH) that has one oxygen atom already inserted into the βTrp57 indole ring (βTrp57-OH of preTTQ) (7), to mature TTQ-containing MADH (8) (Fig. 1).Catalysis by MauG proceeds via a bis-Fe(IV) redox state, which may be generated by reaction of di-Fe(II) MauG with O 2 or di-Fe(III) MauG with H 2 O 2 (9). Catalytically active crystals of the MauG-preMADH protein complex show that the site of TTQ formation on β-preMADH is 40.1 Å from the MauG highspin heme iron...

Mutation of Trp93 of MauG to tyrosine causes loss of bound Ca2+ and alters the kinetic mechanism of tryptophan tryptophylquinone cofactor biosynthesis

“…Completion of TTQ biosynthesis on preMADH could be catalysed by MauG in vitro [10,11] (Figure 1). The order of reactions in this biosynthetic process is cross-link formation between the hydroxylated βTrp 57 and βTrp 108 , a second hydroxylation of βTrp 57 , and oxidation of the quinol MADH (methylamine dehydrogenase) to the quinone state [12]. The process requires three two-electron oxidation reactions.…”

Roles of multiple-proton transfer pathways and proton-coupled electron transfer in the reactivity of the bis -Fe IV state of MauG

“…Mutations of this residue disrupted the position and orientation of Glu113, which in turn altered the water network (29). Mutations of Pro107 as well as a Q103A mutation also increased the susceptibility of the three Met residues to autooxidation (15,29).…”

“…1). Concomitant with this ET is the formation of free-radical intermediates on preMADH that go on to form the TTQ product (15). In the absence of preMADH, the autoreduction of the bis-Fe IV redox state to the diferric state leads to inactivation of MauG (16).…”

Could tyrosine and tryptophan serve multiple roles in biological redox processes?

“…It is well established that multi-step tunnelling, called hopping, is required for functional charge transport in many redox enzymes (examples include ribonucleotide reductase [1][2][3][4][5][6][7][8][9], photosystem II [10][11][12], DNA photolyase [13][14][15][16][17][18][19][20][21], MauG [22][23][24][25] and cytochrome c peroxidase [26,27]). Here, we advance the hypothesis that many such enzymes, most especially those that generate high-potential intermediates during turnover, could be irreversibly damaged if the intermediates are not inactivated in some way.…”