Long-Range Electron Transfer Reactions between Hemes of MauG and Different Forms of Tryptophan Tryptophylquinone of Methylamine Dehydrogenase

Abstract

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. 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 ET from diferrous MauG to oxidized TTQ of MADH exhibits a K d of 10.1 μM and 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 which form TTQ and the five-coordinate oxygen-binding heme is not possible, and that ET requires electron hopping via the six-coordinate heme.Methylamine dehydrogenase (MADH)1 is a 119 kDa heterotetrameric α 2 β 2 protein (1,2) with each β subunit possessing a tryptophan tryptophylquinone (TTQ) (3) protein-derived cofactor (4). TTQ is formed by post-translational modification of two tryptophan residues of the polypeptide chain. For TTQ biosynthesis, two atoms of oxygen are incorporated into the indole ring of βTrp57 and a covalent bond is formed between the indole rings of βTrp57 and βTrp108. TTQ biosynthesis requires the action of MauG (5) which is a 42.3 kDa enzyme containing two c-type hemes (6). MauG exhibits homology to diheme cytochrome c peroxidase (7,8), but displays significant differences in catalytic and redox behavior (9,10). Inactivation of mauG, a gene in the methylamine utilization (mau) gene cluster (11) leads to accumulation of a biosynthetic precursor protein (preMADH) in which βTrp57 is monohydroxylated at C7 position, and the covalent crosslink between βTrp57 and βTrp108 is absent (12,13 His205 and Tyr294 (15). While the order is not known, the overall process of MauG-dependent TTQ biosynthesis requires three sequential two-electron oxidations to catalyze the second hydroxylation of βTrp57, the crosslink formation between βTrp57 and βTrp108, and the oxidation to the quinone state.As TTQ is a two-electron redox cofactor, it may be present in MADH in three different redox states. The fully oxidized quinone exhibits a broad absorbance centered at 440 nm, the one-electron reduced semiquinone exhibits an absorption maximum at 428 nm, and the fully reduced quinol exhibits an absorption maximum at 330 nm (16 The kinetic mechanism of initial two-electron oxidation of preMADH by bis-Fe(IV) MauG was previously characterized (17). The reaction exhibited a random kinetic mechanism, in which the order of addition of [O] and preMADH did not matter. Such a random mechanism is in contrast to that typically...

Mutagenesis of tryptophan199 suggests that hopping is required for MauG-dependent tryptophan tryptophylquinone biosynthesis

“…However, as the diferric/diferrous MauG couple is −201 mV, the overall reaction is thermodynamically favorable with a ΔE m of 303 mV (23,34), and this supports the long-range electron tunneling. This difference in mechanism is also consistent with the previous observation that, for WT MauG (27), this favorable reaction is slower (0.07 s −1 ) than the reaction of preMADH with bis-Fe(IV) MauG (0.8 s −1 ), for which we have estimated a ΔE m of about zero, but which can occur via hopping.…”

“…Transient formation of the quinol was observed early in the steady-state reaction, and bis-Fe(IV) WT MauG can oxidize quinol MADH (15). The steady-state kinetic parameters using WT MauG are K m of 11.1 μM and k cat of 4.2 s −1 (27). W199F/K MauGs were able to catalyze this reaction ( Fig.…”

“…Although not involved in TTQ biosynthesis, a thermodynamically favorable ET reaction from diferrous MauG to quinone MADH, which does not require formation of the bis-Fe(IV) state, has been characterized (27). In this reaction WT MauG exhibited a limiting first-order rate constant of 0.07 s −1 and K d value of 10.1 μM.…”

“…Each reaction was performed in 0.01 M potassium phosphate buffer, pH 7.5 at 25°C. In one reaction varied concentrations of quinol MADH were mixed with 2 μM bis-Fe(IV) MauG that had been generated by stoichiometric addition of H 2 O 2 (27). Reactions were monitored from 366-446 nm to observe the conversion of bis-Fe(IV) MauG (E) to diferric MauG (E 0 ).…”

“…Reactions were monitored from 366-446 nm to observe the conversion of bis-Fe(IV) MauG (E) to diferric MauG (E 0 ). In the other reaction varied concentrations of quinone MADH were mixed with 1.25 μM diferrous MauG under anaerobic conditions (27). The reaction was monitored by the decrease in A 550 , which corresponds to the conversion of diferrous MauG (E) to diferric MauG (E 0 )…”