The tightly bound calcium of MauG is required for tryptophan tryptophylquinone cofactor biosynthesis

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., and 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 Ca ²⁺-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 Ca ²⁺-depleted MauG, full TTQ biosynthesis activity and reactivity toward H₂O₂ were restored, and the spectral properties returned to those of native MauG. Kinetic and equilibrium studies of Ca ²⁺ binding to Ca²⁺-depleted MauG indicated a two-step mechanism. Ca²⁺ initially reversibly binds to Ca²⁺- depleted MauG (K_d = 22.4 μM) and is followed by a relatively slow (k = 1.4×10^-3 s^-1) but highly favorable (K _eq = 4.2) conformational change, yielding an equilibrium dissociation constant K_d,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.