Role of Calcium in Metalloenzymes: Effects of Calcium Removal on the Axial Ligation Geometry and Magnetic Properties of the Catalytic Diheme Center in MauG

Abstract

MauG is a diheme enzyme possessing a five-coordinate high-spin heme with an axial His ligand and a six-coordinate low-spin heme with His-Tyr axial ligation. A Ca2+ ion is linked to the two hemes via hydrogen-bond networks, and the enzyme activity depends on its presence. Removal of Ca2+ alters the EPR signals of each ferric heme such that the intensity of the high-spin heme was decreased and the low-spin heme was significantly broadened. Addition of Ca2+ back to the sample restored the original EPR signals and enzyme activity. The molecular basis for this Ca2+-dependent behavior was studied by magnetic spectroscopy. The results show that in the Ca2+-depleted MauG the high-spin heme was converted to low-spin and the original low-spin heme exhibited a change in relative orientations of its two axial ligands. The properties of these two hemes are each different than in native MauG and are now similar to each other. The EPR spectrum of Ca2+-free MauG appears to describe one set of low-spin ferric heme signals with a large gmax and g-anisotropy and a greatly altered electromagnetic relaxation property. Both EPR and Mössbauer spectroscopic results show that the two hemes are present as unusual highly axial low-spin (HALS)-like hemes in Ca2+-depleted MauG, with a smaller orientation angle between the two axial ligand planes. These findings provide insight into the correlation of the enzyme activity with the orientation of axial heme ligands, and describe a role for the calcium ion in maintaining this structural orientation that is required for activity.

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

“…(i) The 'definition' of the dihaem redox centre of MauG should be expanded to include not only Trp 93 , but also the bound Ca 2 + . The results of the present study together with those of previous studies [16,22,23] indicate that tryptophan at position 93 and bound Ca 2 + must both be present with the two haems in order to efficiently catalyse TTQ biosynthesis. The same is likely to be true for DCCPs with respect to the reactions that they catalyse.…”

“…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].…”

Posttranslational Biosynthesis of the Protein-Derived Cofactor Tryptophan Tryptophylquinone

“…This Ca 2+ does not readily dissociate from MauG; however after Ca 2+ removal by extensive treatment with chelators, MauG is no longer able to catalyze TTQ biosynthesis and exhibits altered absorption, resonance Raman and EPR spectra (41, 47). The circular dichroism spectra of native and Ca 2+ -depleted MauG are essentially the same, consistent with Ca 2+ -induced conformational changes involving domain or loop movements rather than general unfolding or alteration of secondary structure.…”

“…It was shown that in the Ca 2+ -depleted MauG the original high-spin heme is converted to low-spin, and the original low-spin heme exhibits a change in relative orientations of its two axial ligands. EPR and Mössbauer spectroscopic results (47) show that the two hemes are present as unusual highly axial low-spin (HALS)-like hemes (48, 49) in Ca 2+ -depleted MauG, with a smaller orientation angle between the two axial ligand planes. The effects of Ca 2+ -depletion are completely reversible.…”

A simple method to engineer a protein-derived redox cofactor for catalysis

“…Removal of this Ca 2+ by chelators yields a Ca 2+ -depleted MauG which has no TTQ biosynthesis activity [11]. This is due to changes in the diheme site which prevent formation of the bis-Fe IV state [12]. Whereas addition of H 2 O 2 to native MauG generates the bis-Fe IV state, the hemes of Ca 2+ -depleted MauG are not reactive towards H 2 O 2 .…”