Towards resolving the complex paramagnetic NMR spectrum of small laccase: Assignments of resonances to residue specific nuclei

Abstract. Laccases efficiently reduce dioxygen to water in an active site containing a tri-nuclear copper centre (TNC). One reason for its efficiency in catalysis of this complex reaction can be the presence of mobility of active site residues. To probe mobility, NMR spectroscopy is highly suitable. However, several factors complicate the assignment of resonances to active site nuclei in laccases. The paramagnetic nature causes large shifts and line broadening. Furthermore, the presence of slow chemical exchange processes of the imidazole rings of copper ligands result in peak doubling. A third complicating factor is that the enzyme occurs in two states, the native intermediate (NI) and resting oxidized (RO) states, with different paramagnetic properties. The present study aims at resolving the complex paramagnetic NMR spectra of the TNC of Streptomyces coelicolor small laccase (SLAC). With a combination of paramagnetically tailored NMR experiments, all eight His Nδ1 and Hδ1 resonances for the NI state are identified, as well as His Hβ protons for the RO state. With the help of second shell mutagenesis, selective resonances are tentatively assigned to the T2 histidines. This study demonstrates approaches that can be used for sequence specific assignment of the paramagnetic NMR spectra of ligands in the TNC that ultimately may lead to a description of the underlying motions.


shown for the 1 H resonances > 21 ppm but shown for the region 12 to 21 ppm, see Figure 2b). SLAC-T1D is predominantly in the NI state, in which the T2 and the T3 sites are coupled, increasing the electronic relaxation rates of the unpaired electrons and thus reducing the paramagnetic relaxation rates of the nuclear spins. Therefore, it is expected that all eight ligand histidine residues are observable. In the 1 H-15 N HMQC ten resonances are seen, among which three undergo chemical In conclusion, all the eight Hd1 from the coordinating histidines of the TNC in SLAC-T1D for the NI state are identified in the spectral region > 21 ppm and five of them show peak doubling due to slow exchange.      2.3. Second shell mutagenesis to assist assignments. To aid in the assignment of the paramagnetic spectrum, mutagenesis could be employed. However, mutation of histidine ligands is expected to result in loss of copper or at least severe redistribution of unpaired electron density, changing the chemical shifts of all paramagnetically shifted protons. In contrast, mutations in the second coordination sphere, of residues that interact with the coordinating ligands, may have moderate effects 180 on the electron spin density distribution. One such mutant, Y108F, has been reported before (Gupta et al., 2012). Tyr108 interacts with the TNC in two ways, with the T2 site through the water/hydroxide ligand and with the T3 ligand His104 through the hydrogen bonding network involving Asp259 ( Figure   S3a). Asp259 is conserved in all laccases, whereas Tyr108 is conserved in the two-domain laccases ( Figure S3b). Asp259 has been reported to play a role in modulating the proton relay during the oxygen 185 reduction reaction (Quintanar et al., 2005a, p.94) and it may also stabilize the Tyr108-TNC interaction.   Table S2). Also, a new resonance a is observed. The HMQC spectrum in the region > 22 ppm of the 1 H is very similar 205 to that of SLAC-T1D, in agreement with the 1 H WEFT spectrum (Figure 3). Most of the 15 N resonances (3, 4, 5, 9, 11, 12 and 15) are downfield shifted except resonances 6, 13 and 16, which are upfield shifted (Figure 3 and Table S2). The three independent chemical exchange processes that were reported for the TNC of SLAC-T1D involving resonance pairs of 3 -5, 9 -11 and 13 -12 (Dasgupta et al., 2020) are conserved and the rates are not affected by the Y108F mutation (Table S1, Figure 3b 210 and Figure S1c), suggesting that the phenolic -OH group of Y108 is not involved in the chemical exchange process. The chemical shift changes show that the two states represented by 3 -5 and 9 -11, respectively are affected similarly by the Y108F mutation (Figure 3d). In contrast, the two states https://doi.org/10.5194/mr-2020-31

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represented by the resonance pair 13 -12 are affected differently, because the nitrogen chemical shift is downfield shifted for resonance 12 and to upfield shifted for resonance 13 (Figure 3d).
It is proposed that resonances 13 and 16, which are most affected by the Y108F mutation ( Figure 3d), are from the histidine ligands of the T2 copper. Due to the proximity of the T2 copper and strong hydrogen bond with a water or hydroxide ligand, the electron spin density can be expected to be delocalized to the tyrosine ring. The loss of the hydrogen bond between the phenolic -OH group of Tyr108 and the water/hydroxide ligand of the T2 copper can result in redistribution of the electron spin 220 density on the coordinating histidine ligands. Figure 3d shows that the Nd1 of the resonances 13 and 16 have the highest chemical shift perturbation of ~ -16 and -14 ppm respectively. Interestingly, resonance 13 is in an exchange process with resonance 12 (Figure 3b) (Dasgupta et al., 2020) and for the latter resonance the Nd1 exhibits a downfield shift due to the Y108F mutation. In the crystal structure 3cg8 (resolution 2.63 Å), the Nd1 of His102 from the T2 site can have two hydrogen bonding partners, 225 carbonyl oxygen of Asp113 and a water molecule (Figure 4a). Modelling the protons and changing the c2 dihedral angle of His102 to -152º and -94º, hydrogen bonds can be formed between Hd1 -Asp113 CO and Hd1 -H2O respectively. The c2 dihedral change does not break the coordination of His102 Ne2 to the copper (Figure 4b and 4c) and is within the allowed range (-90º to -170º) (Dasgupta et al., 2020). This shows that there can be a conformational exchange of His102 between two states with a 230 hydrogen bond between Hd1 and either Asp113 CO or the nearby H2O molecule. The second shell mutation of Y108F suggests that the exchanging resonances 13 and 12 are from a Hd1 nucleus of one of the two T2 copper histidine ligands. Thus, it is proposed that resonance 13 and 12 are from His102 Hd1 for which the ring exchanges between the two states shown in panels Figure 4b and 4c.
Consequently, resonance 16 can be tentatively assigned to the other T2 copper ligand, His234, being 235 also strongly affected by the Y108F mutation. It does not exhibit chemical exchange at temperatures £ 298 K, in agreement with having a single, hydrogen bond with Asp259 CO (Figure 4a). At higher temperatures ( ³ 303 K) however, exchange with resonance 18 is observed. Whereas the 12/13 pair of resonances shows a difference of less than 3 ppm (Dasgupta et al., 2020) and similar linewidth for both signals, the 16/18 pair shows almost 9 ppm difference in chemical shift and resonance 18 is much 240 broader, indicating a more drastic change in spin density on the proton. In combination with the observation that there are no other hydrogen bond acceptors in the proximity, this suggests that resonance 18 represents the His234 Hd1 in a state in which the hydrogen bond to Asp259 is broken.
In such a state the proton would be prone to exchange with bulk water protons but the TNC is very buried, preventing rapid exchange. Similar situations as for His102 are observed for other histidine 245 ligands in the TNC (Table S4). For example, in the crystal structure 6s0o (resolution 1.8 Å) (Gabdulkhakov et al., 2019) Nd1 of His237 can form a hydrogen bond with Asp114 Od1 or water O540, depending on rotation around c2 ( Figure S5). In the crystal structure 3cg8 the equivalent Asp113 Od1 is moved away from the Nd1 and therefore could not form a hydrogen bond ( Figure S5a). Such exchange processes may well represent the resonances pair 3-5 and 9-11. Second-shell mutations 250 around the respective histidine residues can help to confirm this hypothesis.