Sooim Shin

Kinetic Mechanism for the Initial Steps in MauG-Dependent Tryptophan Tryptophylquinone Biosynthesis

The diheme enzyme MauG catalyzes the biosynthesis of tryptophan tryptophylquinone (TTQ), the protein-derived cofactor of methylamine dehydrogenase (MADH). This process requires the six-electron 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 toward H(2)O(2) and the O(2) analogue CO was essentially the same as that of MauG in the preformed enzyme-substrate complex. The addition of H(2)O(2) to diferric MauG generated a diheme bis-Fe(IV) species [i.e., Fe(IV)=O/Fe(IV)] which formed at a rate of >300 s(-1) and spontaneously returned to the diferric state at a rate of 2 x 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) of < or = 1.5 microM 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 preformed enzyme-substrate complex to generate bis-Fe(IV) MauG followed by reaction with PreMADH. Stopped-flow kinetic studies of the reaction of diferrous MauG with CO yielded a faster major transition with a bimolecular rate constant of 5.4 x 10(5) M(-1) s(-1), and slower transition with a rate of 16 s(-1) which was independent of CO concentration. The same rates were obtained for binding of CO to diferrous 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 toward oxygen in the absence of substrate. This kinetic mechanism is also distinct from that of the homologous diheme cytochrome c peroxidases that require a mixed valence state for activity.

Diradical intermediate within the context of tryptophan tryptophylquinone biosynthesis

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 six-electron 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.

Characterization of electron tunneling and hole hopping reactions between different forms of MauG and methylamine dehydrogenase within a natural protein complex

Respiration, photosynthesis, and metabolism require the transfer of electrons through and between proteins over relatively long distances. It is critical that this electron transfer (ET) occur with specificity to avoid cellular damage, and at a rate that is sufficient to support the biological activity. A multistep hole hopping mechanism could, in principle, enhance the efficiency of long-range ET through proteins as it does in organic semiconductors. To explore this possibility, two different ET reactions that occur over the same distance within the protein complex of the diheme enzyme MauG and different forms of methylamine dehydrogenase (MADH) were subjected to kinetic and thermodynamic analysis. An ET mechanism of single-step direct electron tunneling from diferrous MauG to the quinone form of MADH is consistent with the data. In contrast, the biosynthetic ET from preMADH, which contains incompletely synthesized tryptophan tryptophylquinone, to the bis-Fe(IV) form of MauG is best described by a two-step hole hopping mechanism. Experimentally determined ET distances matched the distances determined from the crystal structure that would be expected for single-step tunneling and multistep hopping. Experimentally determined relative values of electronic coupling (H(AB)) for the two reactions correlated well with the relative H(AB) values predicted from computational analysis of the structure. The rate of the hopping-mediated ET reaction is also 10-fold greater than that of the single-step tunneling reaction despite a smaller overall driving force for the hopping-mediated ET reaction. These data provide insight into how the intervening protein matrix and redox potentials of the electron donor and acceptor determine whether the ET reaction proceeds via single-step tunneling or multistep hopping.

Long-range electron transfer reactions between hemes of MauG and different forms of tryptophan tryptophylquinone of methylamine dehydrogenase.

The diheme enzyme MauG catalyzes the post-translational modification of a precursor protein of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. This six-electron oxidation of preMADH requires long-range electron transfer (ET) as the structure of the MauG-preMADH complex reveals that the shortest distance between the modified residues of preMADH and the nearest heme of MauG is 14.0 A [Jensen, L. M. R., Sanishvili, R., Davidson, V. L., and Wilmot, C. M. (2010) Science 327, 1392-1394]. The kinetics of two ET reactions between MADH and MauG have been analyzed. Interprotein ET from quinol MADH to the high-valent bis-Fe(IV) form of MauG exhibits a K(d) of 11.2 microM and a rate constant of 20 s(-1). ET from diferrous MauG to oxidized TTQ of MADH exhibits a K(d) of 10.1 microM and a rate constant of 0.07 s(-1). These similar K(d) values are much greater than that for the MauG-preMADH complex, indicating that the extent of TTQ maturity rather than its redox state influences complex formation. The difference in rate constants is consistent with a larger driving force for the faster reaction. Analysis of the structure of the MauG-preMADH complex in the context of ET theory and these results suggests that direct electron tunneling between the residues that form TTQ and the five-coordinate oxygen-binding heme is not possible, and that ET requires electron hopping via the six-coordinate heme.

Role of calcium in metalloenzymes: effects of calcium removal on the axial ligation geometry and magnetic properties of the catalytic diheme center in MauG.

MauG is a diheme enzyme possessing a five-coordinate high-spin heme with an axial His ligand and a six-coordinate low-spin heme with His-Tyr axial ligation. A Ca(2+) ion is linked to the two hemes via hydrogen bond networks, and the enzyme activity depends on its presence. Removal of Ca(2+) altered the electron paramagnetic resonance (EPR) signals of each ferric heme such that the intensity of the high-spin heme was decreased and the low-spin heme was significantly broadened. Addition of Ca(2+) back to the sample restored the original EPR signals and enzyme activity. The molecular basis for this Ca(2+)-dependent behavior was studied by magnetic resonance and Mössbauer spectroscopy. The results show that in the Ca(2+)-depleted MauG the high-spin heme was converted to a low-spin heme and the original low-spin heme exhibited a change in the relative orientations of its two axial ligands. The properties of these two hemes are each different than those of the heme in native MauG and are now similar to each other. The EPR spectrum of Ca(2+)-free MauG appears to describe one set of low-spin ferric heme signals with a large g(max) and g anisotropy and a greatly altered spin relaxation property. Both EPR and Mössbauer spectroscopic results show that the two hemes are present as unusual highly rhombic low-spin hemes in Ca(2+)-depleted MauG, with a smaller orientation angle between the two axial ligand planes. These findings provide insight into the correlation of enzyme activity with the orientation of axial heme ligands and describe a role for the calcium ion in maintaining this structural orientation that is required for activity.

Suicide Inactivation of MauG during Reaction with O2 or H2O2 in the Absence of Its Natural Protein Substrate

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.

Effects of the loss of the axial tyrosine ligand of the low-spin heme of MauG on its physical properties and reactivity

MauG catalyzes posttranslational modifications of methylamine dehydrogenase to complete the biosynthesis of its protein‐derived tryptophan tryptophylquinone (TTQ) cofactor. MauG possesses a five‐coordinate high‐spin and a six‐coordinate low‐spin ferric heme, the latter with His‐Tyr ligation. Replacement of this tyrosine with lysine generates a MauG variant with only high‐spin ferric heme and altered spectroscopic and redox properties. Y294K MauG cannot stabilize the bis‐Fe(IV) redox state required for TTQ biosynthesis but instead forms a compound I‐like species on reaction with peroxide. The results clarify the role of Tyr ligation of the five‐coordinate heme in determining the physical and redox properties and reactivity of MauG.

Carboxyl Group of Glu113 Is Required for Stabilization of the Diferrous and Bis-Fe(IV) States of MauG.

The diheme enzyme MauG catalyzes a six-electron oxidation required for post-translational modification of a precursor of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. Crystallographic studies have implicated Glu113 in the formation of the bis-Fe(IV) state of MauG, in which one heme is Fe(IV)═O and the other is Fe(IV) with His-Tyr axial ligation. An E113Q mutation had no effect on the structure of MauG but significantly altered its redox properties. E113Q MauG could not be converted to the diferrous state by reduction with dithionite but was only reduced to a mixed valence Fe(II)/Fe(III) state, which is never observed in wild-type (WT) MauG. Addition of H2O2 to E113Q MauG generated a high valence state that formed more slowly and was less stable than the bis-Fe(IV) state of WT MauG. E113Q MauG exhibited no detectable TTQ biosynthesis activity in a steady-state assay with preMADH as the substrate. It did catalyze the steady-state oxidation of quinol MADH to the quinone, but 1000-fold less efficiently than WT MauG. Addition of H2O2 to a crystal of the E113Q MauG-preMADH complex resulted in partial synthesis of TTQ. Extended exposure of these crystals to H2O2 resulted in hydroxylation of Pro107 in the distal pocket of the high-spin heme. It is concluded that the loss of the carboxylic group of Glu113 disrupts the redox cooperativity between hemes that allows rapid formation of the diferrous state and alters the distribution of high-valence species that participate in charge-resonance stabilization of the bis-Fe(IV) redox state.

Steady-state kinetic mechanism of LodA, a novel cysteine tryptophylquinone-dependent oxidase

LodA is a novel lysine‐ε‐oxidase which possesses a cysteine tryptophylquinone cofactor. It is the first tryptophylquinone enzyme known to function as an oxidase. A steady‐state kinetic analysis shows that LodA obeys a ping‐pong kinetic mechanism with values of k cat of 0.22 ± 0.04 s−1, K lysine of 3.2 ± 0.5 μM and K O2 of 37.2 ± 6.1 μM. The k cat exhibited a pH optimum at 7.5 while k cat/K lysine peaked at 7.0 and remained constant to pH 8.5. Alternative electron acceptors could not effectively substitute for O2 in the reaction. A mechanism for the reductive half reaction of LodA is proposed that is consistent with the ping‐pong kinetics.

Site-Directed Mutagenesis of Gln103 Reveals the Influence of This Residue on the Redox Properties and Stability of MauG

The diheme enzyme MauG catalyzes a six-electron oxidation that is required for the posttranslational modification of a precursor of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived cofactor, tryptophan tryptophylquinone (TTQ). Crystallographic and computational studies have implicated Gln103 in stabilizing the FeIV=O moiety of the bis-FeIV state by hydrogen bonding. The role of Gln103 was probed by site-directed mutagenesis. Q103L and Q103E mutations resulted in no expression and very little expression of the protein, respectively. Q103A MauG exhibited oxidative damage when isolated. Q103N MauG was isolated at levels comparable to that of wild-type MauG and exhibited normal activity in catalyzing the biosynthesis of TTQ from preMADH. The crystal structure of the Q103N MauG–preMADH complex suggests that a water may mediate hydrogen bonding between the shorter Asn103 side chain and the FeIV=O moiety. The Q103N mutation caused the two redox potentials associated with the diferric/diferrous redox couple to become less negative, although the redox cooperativity of the hemes of MauG was retained. Upon addition of H2O2, Q103N MauG exhibits changes in the absorbance spectrum in the Soret and near-IR regions consistent with formation of the bis-FeIV redox state. However, the rate of spontaneous return of the spectrum in the Soret region was 4.5-fold greater for Q103N MauG than for wild-type MauG. In contrast, the rate of spontaneous decay of the absorbance at 950 nm, which is associated with charge-resonance stabilization of the high-valence state, was similar for wild-type MauG and Q103N MauG. This suggests that as a consequence of the mutation a different distribution of resonance structures stabilizes the bis-FeIV state. These results demonstrate that subtle changes in the structure of the side chain of residue 103 can significantly affect the overall protein stability of MauG and alter the redox properties of the hemes.