¹³C ENDOR Reveals that the D1 Polypeptide C-Terminus is Directly Bound to Mn in the Photosystem II Oxygen Evolving Complex

Abstract

Antiferromagnetically coupled Mn^IIIMn^IV dimers have been commonly used to study biological systems that exhibit complex exchange interactions. Such is the case for the oxygen evolving complex (OEC) in photosystem II (PSII), where we have studied whether the C-terminal carboxylate of D1-Ala344 is directly bound to the Mn cluster. To probe these protein-derived carboxylate hyperfine interactions, which give direct bonding information, Q-band (34 GHz) Mims ENDOR was performed on a Mn^IIIMn^IV dimer ([Mn^IIIMn^IV(μ-O)₂μ-OAc(TACN)₂](BPh₄)₂) (1) that was labeled with ¹³C (I = 1/2) at the carboxylate position of the acetate bridge. Ã_dip is computed based on atomic coordinates from available x-ray crystal structures to be [−2.4, −0.8, 3.2] MHz. The value for A_iso was determined based on simulation of the experimental ENDOR data, for complex 1 A_iso = −1 MHz. Similar studies were then performed on PSII from Synechocystis sp. PCC 6803, in which all alanine-derived C=O groups are labeled with ¹³C including the C-terminal α-COO⁻ group of D1 (Ala344), as well as PSII proteins uniformly labeled with ¹³C Using recent x-ray crystallography data from T. elongatus the values for Ã_dip were calculated and simulations of the experimental data led to A_iso values of 1.2, 1, and 2 MHz, respectively. We infer from complex 1 that an A_iso significantly larger than 1.2 MHz for a Mn-coordinating carboxylate moiety is unlikely. Therefore, we support the closer arrangement of Ala344 suggested by the Loll and Guskov structures and conclude that the C-terminal carboxylate of D1 polypeptide is directly bound to the Mn cluster.

Extensive research efforts have focused on characterizing the structure and ligand environment of the [Mn₄Ca] cluster that makes up the catalytic core of the oxygen-evolving complex (OEC) in photosystem II (PSII). The OEC catalyzes the light-induced 4-electron oxidation of two molecules of H₂O to O₂ and 4 H⁺ via a 5-step reaction pathway referred to as the “S state” or “Kok” cycle.¹ When the OEC is poised in the S₂ state, EPR reveals a g = 2 “multiline” signal with ≈19 ⁵⁵Mn hyperfine lines resolved in the X-band CW spectrum.² This signal is attributed to an S = 1/2 ground state arising from antiferromagnetic coupling of the four manganese ions and is reminiscent of EPR spectra of oxido bridged Mn^IIIMn^IV dimers. One such spectroscopic model of the OEC was the subject of a pulse EPR study in which electron nuclear double resonance (ENDOR) and electron spin-echo envelope modulation (ESEEM) spectroscopies probed water and methanol ligation to the Mn dimer core.³ Those results provided a basis for the exploration of water and methanol binding to biological Mn clusters.^3,4

In the present study, ENDOR spectroscopy is used to characterize the interaction of carboxylate ligands with the Mn ions of exchange-coupled clusters. Figure 1A presents the Q-band (34 GHz) Mims ENDOR spectrum of ¹³C (I = 1/2) in the carboxylate position of an acetate bridge between a Mn^III and Mn^IV in [Mn^IIIMn^IV(μ-O)₂μ-OAc(TACN)₂](BPh₄)₂ (1) (TACN = 1,4,7-triazacyclononane). This acetate moiety is invoked as a model for protein-derived carboxylates thought to coordinate the OEC in PSII.⁵ We compare these ¹³C-1 results to ENDOR spectra of PSII from Synechocystis sp. PCC 6803 for which (i) the C-terminal α-COO⁻ group (Ala344) of D1 and all alanine-derived C=O groups are labeled with ¹³C (α-¹³C-Ala PSII; Fig. 1B) and (ii) all C atoms are uniformly labeled with ¹³C (Fig. 1C).

Figure 1.

Q-band Mims ENDOR spectra (black) of (A) ¹³C-1; (B) α-¹³C-Ala PSII; (C) ¹³C-PSII. Experimental parameters: ν_MW = 33.9 GHz; B₀ = 1218–1220 mT; selected τ values from 180 to 300 ns were used (see SI); t(π/2) = 20 ns; t(π_RF) = 64 μs; T = 12 K (¹³C-1) and 5 K (PSII). Simulation (blue) parameters for ¹³C-1 spectrum: Ã_dip = [−2.5, −0.8, 3.2]; A_iso = −1.0 MHz; line-width 0.2 MHz.

The CW EPR spectra of the ¹³C and ¹²C acetate isotopologues of 1 are essentially identical (Fig. S1). While ¹²C-1 gives no ENDOR intensity around the ¹³C Larmor frequency (ν_L(¹³C) = 13.0 MHz at B₀ = 1218.0 mT), the ¹³C-1 spectrum possesses a pair of structured peaks centered at ν_L(¹³C) and split by 1.9 MHz (Fig. 1A). The dipolar (ã_dip) and isotropic (a_iso) hyperfine interactions govern the magnitude of the splitting between the ENDOR peaks. ã_dip is defined as the site-specific through-space interaction of the ¹³C nuclear spin with the electron spin on each Mn ion. a_iso originates from unpaired electron spin population in the valence s-orbital of the ¹³C atom. For multi-metal clusters such as 1, exchange interactions between the metal ions lead to an effective spin (S = 1/2) that is different from the spins of the isolated ions (S_Mn^III = 2 and S_Mn^IV = 3/2). Thus the observed hyperfine (A_iso l̃ + Ã_dip) must be scaled by the appropriate projection factors to give a_iso and ã_dip before deriving both the covalent character of the bond and the distance between the unpaired spin for each site and the magnetic ligand nucleus. The projection factors for the Mn^III and Mn^IV ions in 1 are +2 and −1, respectively.³ The site ã_dip representing each Mn···¹³C interaction was computed using the coordinates from the high-resolution x-ray crystallographic structure.⁶ These two hyperfine contributions were then scaled by the projection factors and transformed into a common frame to give Ã_dip.³ With the effective Ã_dip contribution fixed, the value for A_iso was varied from +4 to −4 MHz until a satisfactory simulation of the experimental data was achieved. To resolve the structure evident in the ENDOR features of all spectra in Figure 1, the simulations required an isotropic line-width parameter no larger than 0.2 MHz.⁷ We also accounted for τ-dependent blind spots inherent to Mims ENDOR spectra.⁸ Using this procedure, the simulation parameters that gave the best fit to the experimental data of ¹³C-1 were Ã_dip = [−2.4, −0.8, 3.2] MHz and A_iso = −1.0 MHz (Fig. 1A). This value for A_iso is small compared to that found for a carbon bound directly to a paramagnetic center,⁹ as the α-C of the acetate is two bonds away from each Mn ion.

Results from biochemical and spectroscopic studies⁵ and those from x-ray crystallography^10–12 identify a number of residues as possible ligands to the Mn ions in the OEC. However, these studies give somewhat conflicting results. For example, D1-Ala344 is positioned differently in each x-ray structure. The Ferreira structure shows the COO⁻ group of the D1 polypeptide bound to the Ca ion while structures provided by Loll and Guskov position it as a ligand to Mn_B (Fig. S2). Fourier transform infrared (FTIR) difference results indicate that D1-Ala344 is not bound to Ca but is instead bound to a Mn ion.¹³ Such discrepancies between crystallographic data in comparison to spectroscopy are perhaps not surprising as the x-rays used to collect the diffraction data cause reduction of all Mn ions to Mn^II and resultant changes to the coordination geometry.¹⁴ Therefore, nondestructive spectroscopic methods like FTIR and EPR must also be utilized to characterize the native ligand environment of the [Mn₄Ca] cluster.

The ENDOR spectrum of α-¹³C-Ala PSII is presented in Fig. 1B. While all alanines are isotopically labeled, only D1-Ala344 is found close enough to the Mn cluster to have a non-negligible hyperfine coupling.¹⁵ This spectrum exhibits a splitting pattern similar to that observed for ¹³C-1. Nuclear coordinates needed to compute Ã_dip for the OEC^···D1-Ala344 interaction were taken from the T. elongatus x-ray structures of Loll, Guskov, and Ferreira. The projection factors used were those reported in ref. 16, assuming that Mn^III is at the Mn_C position (Fig. S2). Our choice for the position of the Mn^III ion is based on multifrequency ESEEM results that suggest D1-His332 is bound to the Mn^III ion in the S₂ state of the OEC.¹⁷ However, spectra using alternate sets of projection factors were also simulated which are consistent with the reported results (see Supporting Information). With Ã_dip fixed for each x-ray structure, the value of A_iso needed to simulate the α-¹³C-Ala PSII data was determined to be 1.2, 1.0, and 2.0 MHz, respectively (Fig. 2). The longer distance between D1-Ala344 and the Mn ions in the Ferreira structure corresponds to a small Ã_dip and thus a larger A_iso must be used to provide the proper peak splitting, and even then the peaks are quite narrow and featureless compared to the experimental spectrum. However, with D1-Ala344 directly bound to Mn in the Loll and Guskov structures, a larger Ã_dip is computed—requiring a smaller A_iso—leading to a better fit of the structural features in Figure 2. Based on our analysis of the ENDOR data of ¹³C-13 which possesses an intimate Mn^···COO⁻ interaction—we regard any value for an A_iso significantly larger than 1.2 MHz for a Mn-coordinating carboxylate moiety as unlikely. Furthermore, we expect an A_iso ≈ 0 MHz for a ligand bound only to the diamagnetic Ca ion, as there would be no unpaired electron spin density at the nucleus.¹⁸ Considering this, we conclude that C-terminal carboxylate of D1-Ala344 is a Mn ligand and cannot be bound to only the Ca ion.

Figure 2.

Q-band Mims ENDOR spectrum of α-¹³C-Ala PSII (black) with simulations (blue) generated using coordinates from selected x-ray data of T. elongatus PSII. Simulation parameters: line-width 0.2 MHz (A) Loll, A_iso = 1.2 MHz, Ã_dip = [−2.2, 0.8, 1.4]; (B) Guskov, A_iso = 1.0 MHz, Ã_dip = [−2.0, −1.5, 3.5]; and (C) Ferreira, A_iso = 2.0 MHz, Ã_dip = [−0.4, −0.2, 0.6].

The ENDOR spectrum of uniformly ¹³C-labeled PSII (Fig. 1C) also shows prominent peaks split by •1.7 MHz, a slightly smaller splitting than observed for the α-¹³C-Ala PSII sample (Fig. 1B). We note that if we scale the ENDOR signals to the fundamental spin echo signal, the intensity of the uniformly labeled sample is appreciably more intense than that for the α-¹³C-Ala sample. This implies that there are multiple ¹³C-containing moieties with couplings in the 1.7–1.9 MHz range. The slightly larger splitting in the α-¹³C-Ala PSII spectrum indicates the OEC Mn interaction with the D1-Ala344 ¹³C is one of the strongest (see red line Fig. 1). An additional set of peaks with a resolved splitting of 0.9 MHz arises from another group of ¹³C nuclei with appreciably weaker couplings. A number of mutagenesis, FTIR difference and x-ray crystallographic studies suggest additional amino-acid derived carboxylate ligands to the OEC (see Fig. S2).^5,10,11 Further ¹³C ENDOR studies are currently underway to identify which of these amino acids are responsible for these more weakly-coupled features.

In conclusion, ¹³C ENDOR data indicate that multiple classes of ¹³C-containing moieties are coupled to the [Mn₄Ca] cluster. In particular, these data conclusively show that the C-terminal carboxylate of D1-Ala344 is bound to at least one Mn ion.