Structural Properties and Catalytic Implications of the SPASM Domain Iron-Sulfur Clusters in Methylorubrum extorquens PqqE

Abstract

Understanding the relationship between the metallocofactor and its protein environment is the key to uncovering the mechanism of metalloenzymes. PqqE, a radical S-adenosylmethionine enzyme in pyrroloquinoline quinone (PQQ) biosynthesis, contains three iron-sulfur cluster binding sites. Two auxiliary iron-sulfur cluster binding sites, designated as AuxI and AuxII, use distinctive ligands compared to other proteins in the family while their functions remain unclear. Here, we investigate the electronic properties of these iron-sulfur clusters and compare the catalytic efficiency of wild-type (WT) Methylorubrum extorquens AM1 PqqE to a range of mutated constructs. Using native mass spectrometry, protein film electrochemistry, and electron paramagnetic resonance spectroscopy, we confirm the previously proposed incorporation of a mixture of [2Fe-2S] and [4Fe-4S] clusters at the AuxI site and are able to assign redox potentials to each of the three iron-sulfur clusters. Significantly, a conservative mutation at AuxI, C268H, shown to selectively incorporate a [4Fe-4S] cluster, catalyzes an enhancement of uncoupled S-adenosylmethionine cleavage relative to WT, together with the elimination of detectable peptide cross-linked product. While a [4Fe-4S] cluster can be tolerated at the AuxI site, the aggregate findings suggest a functional [2Fe-2S] configuration within the AuxI site. PqqE variants with nondestructive ligand replacements at AuxII also show that the reduction potential at this site can be manipulated by changing the electronegativity of the unique aspartate ligand. A number of novel mechanistic features are proposed based on the kinetic and spectroscopic data. Additionally, bioinformatic analyses suggest that the unique ligand environment of PqqE may be relevant to its role in PQQ biosynthesis within an oxygen-dependent biosynthetic pathway.

Figure 1.. Biosynthesis of PQQ in Methylobacterium extorquens AM1.

PQQ biosynthesis requires 7 genes pqqABCDEFG, pqqF/G present outside of the operon. The first step of PQQ biosynthesis is the C-C crosslink of glutamate and tyrosine of PqqA. The reaction is catalyzed by PqqE in the presence of chaperone PqqD, reducing reagent and SAM. The crosslinked PqqA is shown undergoing digestion by proteinase PqqF/G prior to the action of PqqB. By analyzing the activity of substrate analogues, an excised cross-linked Glu-Tyr is proposed to generate oxidation by PqqB, followed by spontaneous cyclization, and an eight-electron oxidation by PqqC finally forming PQQ.

Figure 2.. Crystal structure of PqqE and other SPASM/twitch auxiliary iron-sulfur clusters.

A) Crystal structure of PqqE (PDB 6C8V). The iron-sulfur cluster binding sites are shown in a dashed rectangle to the right. The zoom-in structure shows the ligand environment of each iron-sulfur cluster in PqqE. The structural model of the RS [4Fe-4S] cluster was built by SWISS-MODEL (https://swissmodel.expasy.org/) using CteB (PDB 5WGG) as a structural template. B) The crystal structure of SuiB (PDB 5V1Q) and its [4Fe-4S] AuxI site. C) The crystal structure of SkfB (PDB 6EFN) and its auxiliary [2Fe-2S] cluster.

Figure 3.. Native MS confirms that the as-purified PqqE contains a mixture of iron-sulfur clusters.

A) Mass spectrum showing charge states of PqqE formed in native MS. The 13+ charge state is formed at the highest abundance. B) View of the mass spectrum showing detail for the 13+ charge state. Species are labeled alphabetically and the molecular weights of each species are listed in Table 1.

Figure 4.. Bioinformatic analysis of the iron-sulfur cluster-containing proteins in the PDB that use a CXC motif in iron-sulfur cluster binding.

A) Among 637 [2Fe-2S] cluster-containing entries and 1016 [4Fe-4S] cluster-containing entries in the PDB, CXC motifs were found as ligands for 47 of [2Fe-2S] and 9 of [4Fe-4S] clusters. B) The majority of proteins containing a CXC binding [2Fe-2S] cluster are from aerobes or facultative anaerobes. C) The variation of the middle residue in CXC shows that CRC is found preferentially in [2Fe-2S]. D) The crystal structure of Ralstonia solanacearum CDGSH iron-sulfur protein (PDB 3TBM) shows a CRC motif binding to a [2Fe-2S] cluster using two cysteines to coordinate one iron. E) Crystal structure of E. coli aldehyde oxidase (PDB 5G5G) shows a CRC motif binding to a [2Fe-2S] cluster using two cysteines to coordinate two irons. F) Crystal structure of Methanothermococcus thermolithotrophicus heterodisulfide reductase (PDB 5ODC) shows a CHC motif binding to a [4Fe-4S] cluster using two cysteines to coordinate two irons and the presence of a fifth cysteine near the cluster.

Figure 5.. Cyclic and square-wave voltammetry of PqqE and its cluster knockout variants.

For each protein, the cyclic voltammogram is shown on the left panel and the square-wave voltammogram is shown on the right panel. Measurements and the signal after baseline subtraction are shown in colored solid lines. Baseline measurements are shown in gray. A) WT signal (black) was fitted to three one-electron transfers: AuxII (green dashed line), AuxI (dark red dashed line) and RS (blue dashed line). B) AuxI/AuxII signal (purple) was fitted to two one-electron transfers (dashed line). C) RS/AuxII signal (pink) was fitted to two one-electron transfers (dashed line). D) RS/AuxI signal (magenta) was fitted to two one-electron transfers (dashed line). Reduction potentials obtained from cyclic and square-wave voltammetry of E) AuxI only (brown) and F) RS only (blue) were used for fitting the reduction potentials in single cluster knockouts. Cyclic voltammogram measured with a scan rate of 50 mV/s and square-wave voltammogram measured with a frequency of 15 Hz and amplitude of 50 mV using variants at 4 °C and pH 7.5 on pencil graphite electrode (PGE) modified with multiwall carbon nanotube (MWCNT).

Figure 6.. X-band CW EPR spectra of AuxI only variant.

A) Low-temperature (15 K) X-band EPR spectrum of dithionite-reduced AuxI only variant, which contains only the reduced [2Fe-2S]⁺ cluster with g-values = [2.004, 1.958, 1.904]. Experimental data are shown in black and the simulation is shown in red. B) Low-temperature (10 K) X-band EPR spectrum of Ti(III) citrate-reduced AuxI only variant (blue), in which no g₁ = 2.104 signal corresponding to the reduced [4Fe-4S]⁺ cluster in the AuxI site is observed. The WT PqqE signal (black) obtained under the same condition shows the g₁ = 2.104 signal that corresponds to the reduced [4Fe-4S]⁺ cluster in the AuxI site.

Figure 7.. Cyclic voltammetry of PqqE variants with single ligand replacement.

For each protein, measurements and the signals after baseline subtraction are shown in colored solid lines. Baselines are in gray. Species fittings are in dashed lines. A) RS/AuxI/D319C signal (black) was fitted by three one-electron transfers (dashed line). B) RS/AuxI/D319H signal (cyan) was fitted by three one-electron transfers (dashed line). C) RS/D319C signal (purple) was fitted by two one-electron transfers (dashed line). D) AuxI/D319C signal (wine) was fitted by two one-electron transfers (dashed line). E) RS/C268H/AuxII signal (dark blue) was able to be fitted by two one-electron transfers (dashed line). Cyclic voltammograms were measured with a scan rate of 50 mV/s at 4 °C and pH 7.5 on PGE modified with MWCNT.

Figure 8.. X-band CW EPR spectra of RS/C268H/AuxII variant.

A) Low-temperature (10 K) X-band EPR spectrum of dithionite-reduced RS/C268H/AuxII (black), which contains the corresponding signals from the reduced RS [4Fe-4S]⁺ cluster (g = [2.040, 1.927, 1.897]) and the reduced AuxII [4Fe-4S]⁺ cluster (g = [2.059, 1.940, 1.903]). The simulated signal for RS and AuxII clusters is shown in black. B) High-temperature (60 K) X-band EPR spectrum of dithionite-reduced RS/C268H/AuxII (blue), which shows no [2Fe-2S] cluster signal. C) Low-temperature (10 K) X-band EPR spectrum of Ti(III) citrate-reduced RS/C268H/AuxII (black), which shows the g₁ = 2.104 signal corresponding to the reduced [4Fe-4S]⁺ cluster in the AuxI site. The shaded area is due to the Ti(III) EPR signal.

Figure 9.. Comparison of 5’-dA and crosslinked PqqA production between WT PqqE and its variants.

Activities of WT PqqE (red), reconstituted WT PqqE (orange), RS/AuxI/D319C (green), RS/AuxI/D319H (blue), RS/AuxI (magenta), RS/AuxII (wheat), AuxI/AuxII (cyan), RS only (yellow), and RS/C268H/AuxII (gray), are plotted based on their production of 5’-dA (left panel) and peptide modification (right panel). All the experiments were performed in duplicate. The production of 5’-dA and the crosslinked PqqA is determined after a 16-hour reaction at room temperature in the anaerobic chamber.

Figure 10.. Proposed mechanism of PqqE.

The biological reduction system used in this study includes [2Fe-2S]-ferredoxin (darker gray), ferredoxin-NADP+ reductase (lighter gray), and NADPH (blue spheres). The electron generated from the oxidation of NADPH (blue spheres) by flavin (yellow spheres) is transferred via a ferredoxin [2Fe-2S] center (yellow and orange spheres) to PqqE, which is used for a reductive SAM cleavage. Generation of the 5′-dA radical at the RS site is followed by hydrogen atom abstraction at Glu-16 on PqqA. The glutamyl radical attacks the Tyr-20 and forms a de novo C−C bond in PqqA. This step, coupled to proton transfer, generates a highly reactive tyrosine radical within the Tyr ring, leading to a return of an electron back to the iron−sulfur cluster(s) in PqqE. This electron can be recycled in the next round of the catalysis when new SAM and PqqA/D bind to PqqE. An uncoupling reaction (gray dashed arrows) will occur when this electron is lost, which leads to the need for the exogenous reduction system in the next turnover. The boxed gray areas indicate the location of the proposed electron hopping within the system.