Structural Dynamics of the Methyl-Coenzyme M Reductase Active Site Are Influenced by Coenzyme F430 Modifications

Methyl-coenzyme M reductase (MCR) is a central player in methane biogeochemistry, governing methanogenesis and the anaerobic oxidation of methane (AOM) in methanogens and anaerobic methanotrophs (ANME), respectively. The prosthetic group of MCR is coenzyme F430, a nickel-containing tetrahydrocorphin. Several modified versions of F430 have been discovered, including the 172-methylthio-F430 (mtF430) used by ANME-1 MCR. Here, we employ molecular dynamics (MD) simulations to investigate the active site dynamics of MCR from Methanosarcina acetivorans and ANME-1 when bound to the canonical F430 compared to 172-thioether coenzyme F430 variants and substrates (methyl-coenzyme M and coenzyme B) for methane formation. Our simulations highlight the importance of the Gln to Val substitution in accommodating the 172 methylthio modification in ANME-1 MCR. Modifications at the 172 position disrupt the canonical substrate positioning in M. acetivorans MCR. However, in some replicates, active site reorganization to maintain substrate positioning suggests that the modified F430 variants could be accommodated in a methanogenic MCR. We additionally report the first quantitative estimate of MCR intrinsic electric fields that are pivotal in driving methane formation. Our results suggest that the electric field aligned along the CH3-S-CoM thioether bond facilitates homolytic bond cleavage, coinciding with the proposed catalytic mechanism. Structural perturbations, however, weaken and misalign these electric fields, emphasizing the importance of the active site structure in maintaining their integrity. In conclusion, our results deepen the understanding of MCR active site dynamics, the enzyme’s organizational role in intrinsic electric fields for catalysis, and the interplay between active site structure and electrostatics.


Supporting Methods
Cultivation of M. acetivorans.Methanosarcina acetivorans WWM60 was obtained from Dr. Biswarup Mukhopadhyay (Virginia Tech, originally from W. W. Metcalf, University of Illinois at Urbana-Champaign) and was cultured in high-salt medium 1 with acetate (200 mM sodium acetate), methanol (125 mM), or trimethylamine (50 mM) at 37 ºC.A typical F 430 extraction was performed with cells from 300-500 mL of culture.Bottles with Balch-type closures were used; either 125 mL (Wheaton) bottles containing 75 mL or 1 L (Chemglass) bottles containing 500 mL.The medium was reduced with 0.025% sodium sulfide before inoculation and the headspace contained 80% N 2 /20% CO 2 (10 psi).
Partial purification and analysis of F 430 s.Cells were harvested by centrifugation aerobically and then processed immediately for F 430 extraction.Pellets (~0.2 g wet weight) were resuspended in 4 mL of water followed by sonication on ice using a Misonex sonicator equipped with a microtip.Two sonication cycles, each one minute, were performed with duty cycle set at 50 (%/1 sec) and the power at 4. Formic acid was added to a final concentration of 1% followed by centrifugation of the acidified lysates at 4,400 x g.The supernatant was transferred to a new tube, neutralized with NaOH, and diluted 2x with 50 mM Tris, pH 7.5.The resulting sample was filtered and then applied to a gravity flow column with Q Sepharose Fast Flow resin (2 x 10 cm, Cytiva) equilibrated with 50 mM Tris, pH 7.5.After washing with 10 mL of 50 mM Tris, pH 7.5, the F 430 was eluted with 10 mL of 20 mM formic acid.The sample was concentrated to 500 µL under vacuum at 30 ºC, then applied to a 3 kDa MWCO Amicon Ultra concentrator (Millipore-Sigma) to remove any remaining large molecules.The filtrate was further concentrated down to ~100 µL followed by LC-MS or HPLC with diode array (HPLC-DAD) analysis.
For high-resolution LC-MS analysis, a Waters Synapt G2-S HDMS interfaced with an Acquity I-Class UPLC system with an Acquity BEH C18 column (2.1 mm x 50 mm; particle size, 1.7 m; maintained at 35 ºC) was used.Solvent A was water with 0.1% formic acid, and solvent B was acetonitrile with 0.1% formic acid.The flow rate was 0.2 ml/min, and gradient elution was employed in the following manner (time [min], percent solvent B): (0.01, 1), (5, 20), (7, 95), and (8, 95).Two microliters of sample were injected.The mass spectral data were collected in highresolution MSe continuum mode (nonselective MS/MS acquisition mode).Parameters were a 2.8-kV capillary voltage, a 125°C source temperature, a 350°C desolvation temperature, a 35-V sampling cone, 50-liter/h cone gas flow, a 500-liter/h desolvation gas flow, and a 6-liter/h nebulizer gas flow.The collision energies for the low-energy scans (function 1) were 4 V and 2 V in the trap region and the transfer region, respectively.Collision energies for the high-energy scans (function 2) were ramped from 25 to 45 V in the trap region and 2 V in the transfer region.Data were analyzed using MassLynx software (Waters).
For HPLC-DAD analysis, a Shimadzu a Shimadzu HPLC equipped with a photodiode array (PDA) detector and a Kinetex Polar C18 column (Phenomenex, 2.6 µm, 150 x 4.6 mm) was used.The column oven was set at 30°C.Solvent A was 0.1% (v/v) formic acid in water and solvent B was 100% methanol.The flow rate was 0.7 mL min -1 and the method consisted of 95% A for 3 min followed by a 20 min linear gradient to 70% B, then a 1 min linear to 100% B followed by 100% B for 3 min.Ten microliters of concentrated cell extract were injected.
Flat-bottom restraints applied during alchemical transformation.In order to ensure a gentle accommodation of the modified F 430 cofactors by the MCR active-site residues, we employed an alchemical transformation using flat-bottom restraints on key distances and angles that govern the orientation of cofactors within the active site, as shown in Figure S5.Distances were allowed to fluctuate ±1Å from their crystallographic value before the restraint force started to be applied.Two angles were defined to prevent a tilting motion of the F 430 within the active site: φ was composed by the thioether sulfur atom of CH 3 -S-CoM, the Ni(I) atom of F 430 and the pyrrole nitrogen N1 of F 430 macrocyclic ring, while ψ was composed by the same first two atoms and the pyrrole nitrogen N3 of F 430 .In this way, both angles are almost orthogonal to each other and prevent tilting motions more effectively.
Parameters for electric field calculations.TUPÃ uses a configuration file in which is defined the environment set, probe set and other parameters for specific calculation modes.The configuration file used in this work is shown below, where {X} can be A or B, depending on the active site being analyzed.Segment ID COMA and COMB stand for CH 3 -S-CoM molecules, while protein segment IDs were defined as PROA, PROB, PROC, PROD, PROE and PROF.To account only for the contribution of the hydrophobic cage residues to the electric field, we modified the environment set definition to: Force field parameters for MCR cofactors.The parameters below for CH

Supporting Results
Identification of a modified F 430 in Methanosarcina acetivorans.In previous work, 2 we identified a modified version of F 430 in Methanocaldococcus jannaschii.Based on the exact mass, characteristic fragment ions, and the UV-vis spectrum, we proposed the modification to be a cyclized mercaptopropionate moiety attached as a thioether to the 17 2 position of F 430 .To gain insights into the significance and potential function(s) of modified F 430 s, we have explored the existence of modified F 430 s in other methanogens, including Methanosarcina acetivorans.Interestingly, we identified a modified F 430 in M. acetivorans that is one mass unit less than our previously identified mercaptopropionate-F 430 (1008 vs. 1009, Figure S9A) .This difference corresponds to the replacement of a carboxyl group (45 Da) with a primary amide (44 Da).Notably, the mass spectrum of the newly identified F 430 displays a prominent doubly charged ion (Figure S9A), supporting the assignment of an amide-containing modification compared to a carboxyl group.The mass spectrum reveals the characteristic nickel isotope pattern with an intense [M+2] + peak due to Ni-60 (26% natural abundance) (Figure S9B).The modified F 430 is comprised of four major peaks that elute before the canonical F 430 during reverse-phase HPLC (Figure S9C).The various peaks are expected to be stereoisomers that are likely produced chemically during the processing of the cell extract.UV-vis analysis revealed the characteristic 430 nm absorbance maximum (Figure S9D), and the absorbance spectrum was identical to that of the unmodified F 430 in these cells.MS/MS fragmentation shows a major fragment where the modification is cleaved off to yield the parent F 430 (m/z 905), supporting a structure where a single new side-chain to F 430 has been installed.Based our current spectral data and assuming that this new F 430 modification is related to our previously reported F 430 modification, 2 we propose a possible structure with a 3mercaptopropanamide modification where the sulfur is inserted into a C-H bond.We propose that the modification occurs at the 17 2 position since this is one of the few sites that would produce the 905 fragment ion and the F-ring appears to be a hotspot for F 430 modifications. 3,4 owever, future detailed structure determination experiments will be required to elucidate the true structure.Interestingly, the modified F 430 was only identified in M. acetivorans cultures grown on acetate and was not present when the organism was grown on methanol or trimethylamine.Although the amount of the modified F 430 in acetate-grown M. acetivorans varies substantially between different preparations, we have observed the modified version existing at amounts up to ~40% of the total F 430 .

[
Environment Selection] # The atoms from which we calculate the electric field sele_environment = segid PRO* or segid F43{X} or segid COB{X} [Probe Selection] # Provide the probe selection for the MODE of your choice # e.g. if bond is used, then selbond1 and selbond2 must be defined.frames written in your trajectory (in picosecond) sele_environment = (segid PRO{X} and (resid 463 or resid 343 or resid 346)) or (segid PRO{Y} and (resid 359 or resid 365)) #M.acetivorans for M.acetivorans MCR, in which {X} and {Y} can be A and C for active site A, or B and D for active site B. For ANME-1 MCR, we used the selection below: sele_environment = (segid PRO{W} and (resid 462 or resid 344 or resid 347)) or (segid PRO{Z} and (resid 357 or resid 363)) # ANME-1

Figure S4 .
Figure S4.Time series of RMSD values of F 430 cofactors in active sites A and B for all replicates of each system.Systems Ma-F 430 , Ma-mtF 430 , and Ma-mpaF 430 are shown in panels A, B and C, respectively, while systems ANME-mtF 430 and ANME-F 430 are shown in panels D and E.

Figure S5 .
Figure S5.Alternative pose of CH 3 -S-CoM in Ma-F 430 system.Time series of key distances describing the CH 3 -S-CoM alternative pose.D1 reflects the distance between the closest sulfonate oxygen to the Ni(I) atom, while D2 shows the distance between the methyl group of CH 3 -S-CoM and the thiol sulfur atom of HS-CoB.

Figure S6 .
Figure S6.Flexibility of CH 3 -S-CoM in the alternative pose.Superposition of conformations sampled by CH 3 -S-CoM in its alternative binding pose for systems Ma-mpaF 430 (A) and Ma-F 430 (B) highlighting the high mobility of the methylthio group during the simulations.F 430 cofactors of each system are shown as reference with carbon atoms colored in green, while Ni(I) atoms are shown as pink spheres.

Figure S7 .
Figure S7.Time series of the effective electric field magnitude (|E eff |) values calculated at the thioether S-CH 3 bond for all systems.Systems Ma-F 430 , Ma-mtF 430 , and Ma-mpaF 430 are shown in panels A, B and C, respectively, while systems ANME-mtF 430 and ANME-F 430 are shown in panels D and E.

Figure S8 .
Figure S8.Flat-bottom restraint scheme applied in this work.A) Restrained distances between cofactors and protein residues in MCR.B) Restrained angles φ and ψ composed by thioether sulfur, Ni(I) and pyrrole nitrogen N1 and N3, respectively.

Figure S9 .
Figure S9.Identification of a modified F 430 in M. acetivorans.(A) mass spectrum showing M + molecular ion at 1008.3472 as well as the prominent doubly charged ion.(B) mass spectrum showing isotope peaks for molecular ion with characteristic nickel isotope peak at [M+2] + .(C) HPLC-DAD analysis with 430 nm extracted chromatogram shown.(D) absorbance spectrum of the modified F 430 .(E) a proposed structure of the modified F 430 .
3 -S-CoM, HS-CoB and F 430 cofactors were obtained from the CGenFF force field version 4.6.