MauG is a di-heme enzyme responsible for the posttranslational modification of two tryptophan residues to form the tryptophan tryptophylquinone cofactor (TTQ) of methylamine dehydrogenase (MADH). MauG converts preMADH, containing monohydroxylated-βTrp57, to fully functional MADH by catalyzing the insertion of a second oxygen atom into the indole ring and covalently linking βTrp57 to βTrp108. Here we report the 2.1 Å resolution X-ray crystal structure of MauG complexed with preMADH. The c-type heme irons and the nascent TTQ site are separated by long distances over which electron transfer must occur to achieve catalysis. In addition one of the hemes has an atypical His-Tyr axial ligation. The crystalline protein complex is catalytically competent, as on addition of hydrogen peroxide MauG-dependent TTQ synthesis occurs.

The diheme enzyme MauG catalyzes the posttranslational modification of the precursor protein of methylamine dehydrogenase (preMADH) to complete biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. Catalysis proceeds through a high valent bis-Fe(IV) redox state and requires long-range electron transfer (ET), as the distance between the modified residues of pre-MADH and the nearest heme iron of MauG is 19.4 Å. Trp199 of MauG resides at the MauG-preMADH interface, positioned midway between the residues that are modified and the nearest heme. W199F and W199K mutations did not affect the spectroscopic and redox properties of MauG, or its ability to stabilize the bis-Fe(IV) state. Crystal structures of complexes of W199F/K MauG with pre-MADH showed no significant perturbation of the MauG-preMADH structure or protein interface. However, neither MauG variant was able to synthesize TTQ from preMADH. In contrast, an ET reaction from diferrous MauG to quinone MADH, which does not require the bis-Fe(IV) intermediate, was minimally affected by the W199F/K mutations. W199F/K MauGs were able to oxidize quinol MADH to form TTQ, the putative final two-electron oxidation of the biosynthetic process, but with k cat ∕K m values approximately 10% that of wild-type MauG. The differential effects of the W199F/K mutations on these three different reactions are explained by a critical role for Trp199 in mediating multistep hopping from preMADH to bis-Fe(IV) MauG during the long-range ET that is required for TTQ biosynthesis.cytochrome | electron hopping | peroxidase | protein oxidation | protein radical L ong-range electron transfer (ET) through proteins is required for biological processes including respiration, photosynthesis, and metabolism. Mechanisms by which ET occurs over large distances to specific sites within a protein have been extensively studied (1-4). For interprotein ET, kinetic mechanisms are more complex, as the overall redox reaction requires additional steps such as protein-protein association and reorientation of the protein complex to optimize the system for ET (5, 6). "Long-range catalysis" is a related process in which the redox center that provides the oxidizing or reducing power is physically distinct from the site of chemical reaction of the substrate, so that long-range ET is required for catalysis. Thus far two enzymes have been postulated to employ long-range catalysis. Ribonucleotide reductase (RNR) catalyzes the formation of deoxyribonucleotides from ribonucleotides by long-range ET via multiple tyrosyl residues (7,8). DNA photolyase is a flavoprotein that catalyzes DNA repair of pyrimidine-pyrimidine dimers via multiple tryptophan residues (9). In these enzymes it is believed that the long-range ET proceeds by hopping (10) through residues that can stabilize a radical state, rather than via a single long-range electron tunneling event.

The diheme enzyme MauG catalyzes the posttranslational modification of a precursor protein of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. It catalyzes three sequential two-electron oxidation reactions which proceed through a high valent bis-Fe(IV) redox state. Tyr294, the unusual distal axial ligand of one c-type heme, was mutated to His and the crystal structure of Y294H MauG in complex with preMADH reveals that this heme now has His-His axial ligation. Y294H MauG is able to interact with preMADH and participate in inter-protein electron transfer, but it is unable to catalyze the TTQ biosynthesis reactions that require the bis-Fe(IV) state. This mutation not only affects the redox properties of the six-coordinate heme but also the redox and CO-binding properties of the five-coordinate heme, despite the 21 Å separation of the heme iron centers. This highlights the communication between the hemes which in wild-type MauG behave as a single diheme unit. Spectroscopic data suggest that Y294H MauG can stabilize a high valent redox state equivalent to Fe(V), but it appears to be an Fe(IV)=O/π radical at the five-coordinate heme rather than the bis-Fe(IV) state. This compound I-like intermediate does not catalyze TTQ biosynthesis, demonstrating that the bis-Fe(IV) state, which is stabilized by Tyr294, is specifically required for this reaction. The TTQ biosynthetic reactions catalyzed by wild-type MauG do not occur via direct contact with the Fe(IV)=O heme, but via long range electron transfer through the six-coordinate heme. Thus, a critical feature of the bis-Fe(IV) species may be that it shortens the electron transfer distance from preMADH to a high valent heme iron.

The diheme enzyme MauG catalyzes a six-electron oxidation required for posttranslational modification of a precursor of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. Crystallographic studies had shown that Pro107, which resides in the distal pocket of the high-spin heme of MauG, changes conformation upon binding of CO or NO to the heme iron. In this study, Pro107 was converted to Cys, Val and Ser by site-directed mutagenesis. The structures of each of these MauG mutant proteins in complex with preMADH were determined, as were their physical and catalytic properties. P107C MauG was inactive and the crystal structure revealed that Cys107 had been oxidatively modified to a sulfinic acid. Mass spectrometry revealed that this modification was present prior to crystallization. P107V MauG exhibited spectroscopic and catalytic properties that were similar to wild-type MauG, but P107V MauG was more susceptible to oxidative damage. The P107S mutation caused a structural change which resulted in the five-coordinate high-spin heme being converted to a six-coordinate heme with a distal axial ligand provided by Glu113. EPR and resonance Raman spectroscopy revealed this heme remained high-spin but with much increased rhombicity as compared to the axial signal of wild-type MauG. P107S MauG was resistant to reduction by dithionite and reaction with H2O2, and unable to catalyze TTQ biosynthesis. These results show that the presence of Pro107 is critical in maintaining the proper structure of the distal heme pocket of the high-spin heme of MauG, enabling exogenous ligands to bind and directing the reactivity of the heme-activated oxygen during catalysis, thus minimizing the oxidation of other residues of MauG.

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...

The diheme enzyme MauG catalyzes a six-electron oxidation required for posttranslational modification of a precursor of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. The crystal structure of the MauG-preMADH complex revealed the presence of a Ca2+ in proximity to the two hemes [Jensen, L.M.R., Sanishvili, R., Davidson, V.L. & Wilmot, C.M. (2010) Science 327, 1392–1394]. This Ca2+ did not readily dissociate; however after extensive treatment with EGTA or EDTA MauG was no longer able to catalyze TTQ biosynthesis and exhibited altered absorption and resonance Raman spectra. The changes in spectral features are consistent with Ca2+-dependent changes in heme spin-state and conformation. Addition of H2O2 to the Ca2+-depleted MauG did not yield spectral changes characteristic of formation of the bis-Fe(IV) state which is stabilized in native MauG. After addition of Ca2+ to the Ca2+-depleted MauG, full TTQ biosynthesis activity and reactivity towards H2O2 was restored, and the spectral properties returned to those of native MauG. Kinetic and equilibrium studies of Ca2+ binding to Ca2+-depleted MauG indicated a two-step mechanism. Ca2+ initially reversibly binds to Ca2+-depleted MauG (Kd = 22.4 μM) and is followed by a relatively slow (k = 1.4 × 10−3 s−1) but highly favorable (Keq = 4.2) conformational change, yielding an apparent equilibrium Kd,eq value of 5.3 μM. The circular dichroism spectra of native and Ca2+-depleted MauG were essentially the same, consistent with Ca2+-induced conformational changes involving domain or loop movements rather than general unfolding or alteration of secondary structure. These results are discussed in the context of the structures of MauG and heme-containing peroxidases.

Biosynthesis of the tryptophan tryptophylquinone (TTQ) cofactor activates the enzyme methylamine dehydrogenase. The diheme enzyme MauG catalyzes O-atom insertion and cross-linking of two Trp residues to complete TTQ synthesis. Solution optical and Mössbauer spectroscopic studies have indicated that the reactive form of MauG during turnover is an unusual bisFe(IV) intermediate, which has been formulated as a His-ligated ferryl heme [Fe(IV)=O] (heme A), and an Fe(IV) heme with an atypical His/Tyr ligation (heme B). In this study, Fe K-edge X-ray absorption spectroscopy and extended X-ray absorption fine structure studies have been combined with density functional theory (DFT) and time-dependent DFT methods to solve the geometric and electronic structures of each heme site in the MauG bisFe(IV) redox state. The ferryl heme site (heme A) is compared with the well-characterized compound I intermediate of cytochrome c peroxidase. Heme B is unprecedented in biology, and is shown to have a six-coordinate, S = 1 environment, with a short (1.85-Å) Fe–O(Tyr) bond. Experimentally calibrated DFT calculations are used to reveal a strong covalent interaction between the Fe and the O(Tyr) ligand of heme B in the high-valence form. A large change in the Fe–O(Tyr) bond distance on going from Fe(II) (2.02 Å) to Fe(III) (1.89 Å) to Fe(IV) (1.85 Å) signifies increasing localization of spin density on the tyrosinate ligand upon sequential oxidation of heme B to Fe(IV). As such, O(Tyr) plays an active role in attaining and stabilizing the MauG bisFe(IV) redox state.

MauG catalyzes posttranslational modifications of a methylamine dehydrogenase precursor (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. Trp199 is present at the site of interaction between MauG and preMADH and is critical to this process as it mediates hole hopping during the inter-protein electron transfer that is required for catalysis. Trp199 was converted to Glu and the structure and reactivity of the W199E/preMADH complex were characterized. The results reveal that the nature of residue 199 is also important for productive complex formation between preMADH and MauG.