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 (Kd = 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 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.