Surface Induced Dissociation Coupled with High Resolution Mass Spectrometry Unveils Heterogeneity of a 211 kDa Multicopper Oxidase Protein Complex

Abstract

Manganese oxidation is an important biogeochemical process that is largely regulated by bacteria through enzymatic reactions. However, the detailed mechanism is poorly understood due to challenges in isolating and characterizing these unknown enzymes. A manganese oxidase, Mnx, from Bacillus sp. PL-12 has been successfully overexpressed in active form as a protein complex with a molecular mass of 211 kDa. We have recently used surface induced dissociation (SID) and ion mobility-mass spectrometry (IM-MS) to release and detect folded subcomplexes for determining subunit connectivity and quaternary structure. The data from the native mass spectrometry experiments led to a plausible structural model of this multicopper oxidase, which has been difficult to study by conventional structural biology methods. It was also revealed that each Mnx subunit binds a variable number of copper ions. Becasue of the heterogeneity of the protein and limited mass resolution, ambiguities in assigning some of the observed peaks remained as a barrier to fully understanding the role of metals and potential unknown ligands in Mnx. In this study, we performed SID in a modified Fourier transform-ion cyclotron resonance (FTICR) mass spectrometer. The high mass accuracy and resolution offered by FTICR unveiled unexpected artificial modifications on the protein that had been previously thought to be iron bound species based on lower resolution spectra. Additionally, isotopically resolved spectra of the released subcomplexes revealed the metal binding stoichiometry at different structural levels. This method holds great potential for in-depth characterization of metalloproteins and protein-ligand complexes. Graphical Abstract ᅟ.

Higher-order structural characterisation of native proteins and complexes by top-down mass spectrometry

“…22 SID was also used to localise noncovalent ligands in multimeric complexes. 50,87,88 However, care must be taken to minimize structural rearrangement before SID. Harsh conditions in the source or transfer optics for achieving the best mass resolution could over-activate the complexes and change their shape signicantly, which is measurable by ion mobility.…”

“…SID experiments of these complexes were quick, with data acquisition typically on the order of minutes to hours, and provided critical information to dene their quaternary structures. 50,73,88,91 Fig. 2a illustrates how the data from complex-up MS were recently used to study the previously uncharacterised heterocomplex Mnx, which consists of three proteins: MnxE (12.2 kDa), MnxF (11.2 kDa), and MnxG (138 kDa).…”

“…Unlike SID, CID experiments were not able to faithfully capture the metalbinding properties of MnxF, as, during monomer unfolding, the weakly bound Cu in this subunit was lost. 50,88 Recently, SID was also applied to characterise the subunit arrangement of a plant pseudoenzyme-enzyme hetero-complex between PDX1.2 and PDX1.3 in Arabidopsis. 92 The pseudoenzyme PDX1.2 lost its activity due to mutation of a few key residues at the active site but is nearly identical structurally to the active enzyme PDX1.3.…”

Surface-Induced Dissociation of Noncovalent Protein Complexes in an Extended Mass Range Orbitrap Mass Spectrometer

“…30,31 These features make SID a useful tool to probe biomolecular complexes without solved structures. [32][33][34] Until recently, work involving SID of protein complexes has largely been carried out on TOF platforms. 35,36 With growing utilization of native MS for structural characterization of protein complexes, it has become apparent that greater mass resolution is often desirable and allows for a deeper insight into a given biological system.…”

Surface-Induced Dissociation of Noncovalent Protein Complexes in an Extended Mass Range Orbitrap Mass Spectrometer

“…30,31 These features make SID a useful tool to probe biomolecular complexes without solved structures. [32][33][34] Until recently, work involving SID of protein complexes has largely been carried out on TOF platforms. 35,36 With growing utilization of native MS for structural characterization of protein complexes, it has become apparent that greater mass resolution is often desirable and allows for a deeper insight into a given biological system.…”