Spin Density and Coenzyme M Coordination Geometry of the ox1 Form of Methyl-Coenzyme M Reductase:  A Pulse EPR Study

Abstract

Methyl-coenzyme M reductase (MCR) catalyses the reduction of methyl-coenzyme M (CH3-S-CoM) with coenzyme B (H-S-CoB) to CH4 and CoM-S-S-CoB in methanogenic archaea. Here we present a pulse EPR study of the "ready" form MCR(ox1), providing a detailed description of the spin density and the coordination of coenzyme M (CoM) to the Ni cofactor F430. To achieve this, MCR was purified from cells grown in a 61Ni enriched medium and samples were prepared in D2O with the substrate analogue CoM either deuterated in the beta-position or with 33S in the thiol group. To obtain the magnetic parameters ENDOR and HYSCORE measurements were done at X- and Q-band, and CW EPR, at X- and W-band. The hyperfine couplings of the beta-protons of CoM indicate that the nickel to beta-proton distances in MCR(ox1) are very similar to those in Ni(II)-MCR(ox1-silent), and thus the position of CoM relative to F430 is very similar in both species. Our thiolate sulfur and nickel EPR data prove a Ni-S coordination, with an unpaired spin density on the sulfur of 7 +/- 3%. These results highlight the redox-active or noninnocent nature of the sulfur ligand on the oxidation state. Assuming that MCR(ox1) is oxidized relative to the Ni(II) species, the complex is formally best described as a Ni(III) (d7) thiolate in resonance with a thiyl radical/high-spin Ni(II) complex, Ni(III)-(-)SR <--> Ni(II)-*SR.

Coordination and binding geometry of methyl-coenzyme M in the red1m state of methyl-coenzyme M reductase

“…In MCR red1m (Fig. 6b) [54,62]. In all three cases, the dipolar part of the HI is similar, indicating similar distances of the b-CH 2 group from the nickel.…”

“…At the ''single-crystal'' observer position corresponding to g 3 (Fig. 2c, 1,098 mT) [53], MCR BPS [14,15], and MCR ox1 [54], but vastly different from those of MCR red2r [53], where the four F 430 hydropyrrolic nitrogen hyperfine interactions span a large range, indicating a significant asymmetry in the spin-density distribution (this species has a strong proximal sulfur ligand from HS-CoM). The outcome for MCR red1m is that the spin-density distribution on F 430 is not significantly perturbed by the weak axial ligand.…”

Direct Demonstration of the Presence of Coordinated Sulfate in the Reaction Pathway of Arabidopsis thaliana Sulfite Oxidase Using 33S Labeling and ESEEM Spectroscopy

“…31,32 These works demonstrate that with appropriate instrumentation and suitable choice of the mw frequency, the detection of 33 S is no more complicated than that of other biologically important quadrupolar nuclei ( 2 H, 14 Two-pulse ESE field-sweep spectrum. Experimental conditions: ν mw = 29.458 GHz; time interval between the mw pulses, t = 250 ns; mw pulses, 2×13 ns; temperature, 20 K. Amplitude FTs of two-pulse ESEEM traces recorded for lpH 33 S-At-SO (solid traces) and 32 S-At-SO (dashed traces) at the EPR turning points, as indicated by the labels "g x ", "g y " and "g z ". The bottom trace is the amplitude spectrum of the two-pulse FI ESEEM.…”

“…Direct detection of coordinated sulfate by EPR is not possible for naturally abundant sulfur (94.93% 32 S, I = 0). However, reaction of At-SO with sulfite labeled with 33 S (I = 3/2) should result in a hyperfine interaction (hfi) of Mo(V) with 33 33 SO 3 species is produced and "trapped".…”

Overview of ligand versus metal centered redox reactions in tetraaza macrocyclic complexes of nickel with a focus on electron paramagnetic resonance studies

“…[21][22][23] MCR catalyzes the final step in methane generation by archaea, a process by which most of biogenic methane is created. 22,[24][25][26] MCR is found in several microorganisms, of which that from Methanothermobacter marburgensis is the best characterized (the taxonomy of these organisms is complicated and has been changed over the years; older papers on MCR refer to this organism as Methanobacterium thermoautotrophicum strain Marburg). MCR contains at the active site a prosthetic group comprising a unique macrocyclic ligand, known as F 430 (based on its maximum absorption wavelength), a diagram of which is shown below.…”