The Tightly Bound Calcium of MauG Is Required for Tryptophan Tryptophylquinone Cofactor Biosynthesis

Abstract

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.

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

“…These data suggest a conformational change of the thioether bridge and in turn the orientation of the haem plane. In the spectrum of Ca 2 + -depleted WT MauG, this band shifted to 408 cm − 1 [22]. These features of the low-frequency region of the resonance Raman spectrum of diferric W93Y MauG, which distinguish it from WT MauG, are very similar to, but not identical with, those reported previously in the spectrum of Ca 2 + -depleted MauG.…”

“…Furthermore, this spectral change occurred slowly over several minutes and the spectrum did not spontaneously return to that of the diferric state. These changes in the spectrum of W93Y MauG are very similar to those observed on addition of H 2 O 2 to Ca 2 + -depleted WT MauG [22] (Figure 5C). …”

“…The molar absorption coefficient of W93Y MauG was determined by the pyridine haemochrome method [29] to be ε 409 = 350 000 M − 1 · cm − 1 compared with 309 000 M − 1 · cm − 1 for WT MauG. The changes in absorption maximum, intensity and shape of the Soret peak that were caused by the W93Y mutation are very similar to those caused by removal of Ca 2 + from WT MauG [22] ( Figure 3B). In contrast with the diferric spectra, the absorption spectra of the diferrous WT MauG, W93Y MauG and Ca 2 + -depleted MauG are very similar ( Figures 3C-3D).…”

“…In the spectrum of W93Y MauG, only low-spin haems are present, as is evident by the single ν 3 band at 1507 cm − 1 and absence of a band corresponding to the one at 1478 cm − 1 that is present in WT MauG. Similarly, the resonance Raman spectrum of Ca 2 + -depleted WT MauG displayed only a single ν 3 band at 1506 cm − 1 [22]. The bands in the low-frequency regions of the spectra ( Figure 4B) originate from the in-plane and out-of-plane deformation modes of the porphyrin ring as well as the vibration modes from the haem substituents [31].…”

“…This Ca 2 + does not readily dissociate, but was removed by treatment with chelators. The Ca 2 + -depleted MauG was not able to catalyse TTQ biosynthesis and exhibited Ca 2 + -dependent spectroscopic changes [22,23]. Trp 93 does not interact directly with the Ca 2 + , but forms a hydrogen bond with Asn 66 , one of ligands of Ca 2 + [8].…”