Functional Importance of Tyrosine 294 and the Catalytic Selectivity for the Bis-Fe(IV) State of MauG Revealed by Replacement of This Axial Heme Ligand with Histidine,

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. 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.

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

“…3 E and F). Although this additional absorbance feature is weak, its appearance is consistent with the presence of compound I (18)(19)(20)(21). A similar change in kinetic mechanisms was observed when monitoring the reaction in the NIR region.…”

Tryptophan-mediated charge-resonance stabilization in the bis -Fe(IV) redox state of MauG

“…2A). These spectral changes were more pronounced with the optimized 10:1 ratio of H 2 O 2 than with the stoichiometric ratio used in prior studies (15,16), consistent with a more complete formation of the bis-Fe(IV) species. The absorption maxima for the newly developed Q-bands are similar to those observed in ferryl species from nonglobin peroxidases, such as cytochrome c peroxidase (21) and horseradish peroxidase (22), but are different from those reported for ferryl species from globins (540-545 and 580-590 nm) (23).…”

“…The NIR region of the electronic absorption spectrum of the bis-Fe (IV) species was not explored in previous studies (13,15,16). Inspection of this region revealed that a broad band centered at 950 nm developed upon addition of H 2 O 2 to diferric MauG concomitant with the previously observed changes in the visible region of the spectrum ( Fig.…”

“…Tyr294 is one of the axial ligands of Heme 6C (10). In Y294H MauG, Heme 6C has an axial bis-histidine coordination, and the bis-Fe(IV) intermediate is not generated (16). Instead, this mutant stabilizes a compound I-like species with a characteristic absorption band at 655 nm after H 2 O 2 treatment (16).…”

“…PreMADH was expressed in Rhodobacter sphaeroides. These proteins were purified as described previously (9,13,16,25,26).…”