Proline 107 Is a Major Determinant in Maintaining the Structure of the Distal Pocket and Reactivity of the High-Spin Heme of MauG

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. Crystallographic studies had shown that Pro107, which resides in the distal pocket of the high-spin heme of MauG, changes conformation upon binding of CO or NO to the heme iron. In this study, Pro107 was converted to Cys, Val and Ser by site-directed mutagenesis. The structures of each of these MauG mutant proteins in complex with preMADH were determined, as were their physical and catalytic properties. P107C MauG was inactive and the crystal structure revealed that Cys107 had been oxidatively modified to a sulfinic acid. Mass spectrometry revealed that this modification was present prior to crystallization. P107V MauG exhibited spectroscopic and catalytic properties that were similar to wild-type MauG, but P107V MauG was more susceptible to oxidative damage. The P107S mutation caused a structural change which resulted in the five-coordinate high-spin heme being converted to a six-coordinate heme with a distal axial ligand provided by Glu113. EPR and resonance Raman spectroscopy revealed this heme remained high-spin but with much increased rhombicity as compared to the axial signal of wild-type MauG. P107S MauG was resistant to reduction by dithionite and reaction with H2O2, and unable to catalyze TTQ biosynthesis. These results show that the presence of Pro107 is critical in maintaining the proper structure of the distal heme pocket of the high-spin heme of MauG, enabling exogenous ligands to bind and directing the reactivity of the heme-activated oxygen during catalysis, thus minimizing the oxidation of other residues of MauG.

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

“…3E). Both variants were shown to be capable of stabilizing the bis-Fe(IV) species (25,26). In contrast, the NIR spectral feature was not observed in Y294H MauG upon addition of H 2 O 2 ( Fig.…”

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

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

“…The bis-Fe IV state of MauG describes an alternative strategy by which to stabilize a high-valent heme iron. Several amino acid residues in the proximity of the two hemes have also been implicated in facilitating the formation and stabilizing the bis-Fe IV state (21,(28)(29)(30)(31)(32). The concept of CR stabilization of this high-valent state led to the description of it actually comprising an ensemble of resonance structures including the more traditional high-valent heme species, but with the true bis-Fe IV state as the dominant species (6, 9) (Fig.…”

“…Pro107 which also resides in the heme pocket was also shown to be important. Mutations of this residue disrupted the position and orientation of Glu113, which in turn altered the water network (29). Mutations of Pro107 as well as a Q103A mutation also increased the susceptibility of the three Met residues to autooxidation (15,29).…”

Gates and Binding Pockets for Nitric Oxide with Cytochrome c′, According to Molecular Dynamics

“…Such studies with biologically relevant diatomic gases have been carried out both experimentally and theoretically with various proteins. Experimentally, by i) mapping the location of Xe-atoms on pressurizing the protein with Xe gas [5], implicitly assuming that the binding sites for Xe-atoms are the same as for diatomic gases, because of similar steric bulk and polarity, ii) locating the preferred channels by using molecules that elongate on the channels that connect the protein-active center to the solvent medium [6], iii) using protein mutants to verify the relevance of key residues as to the displacement of the gaseous molecules [7], and iv) detecting conformational changes for close-lying residues on formation of the complex between the protein and the diatomic gas [8]. Theoretical approaches include PMF [9] and 3D-RISM calculations [10], as well as classical MD [11] and biased MD, such as TAMD [12], and RAMD [13].…”