Modulating the cobalt redox potential through imidazole hydrogen bonding interactions in a supramolecular biomimetic protein-cofactor model

This paper describes a supramolecular biomimetic model of the “His-on” configuration and the charge relay system present in certain types of B12-dependent enzymes.

The vitamin B 12 ("B 12 ") cofactors methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl) represent organometallic Co IIIcorrinoids ( Fig. 1 le) that catalyze biologically important methyl transfer and difficult [1,2] rearrangement reactions. 1 In MeCbl-dependent enzymatic reactions, the cobalt-carbon bond of MeCbl is cleaved heterolytically, which generates a square planar Cob(I)alamin species. Alternatively, in the case of AdoCbl-dependent enzymes, homolytic bond scission leads to a protein bound cofactor in the Co II state and an organic 5 0 -deoxyadenosyl radical (Adoc). This difference in organometallic reactivity is remarkable, considering that MeCbl and AdoCbl cofactors share identical features, as (i) the redox-active cobalt ion, (ii) the equatorially chelating corrin ligand, (iii) the lower coordinating dimethylbenzimidazole (Dmbz) base and (iv) the upper cobalt-carbon bond (Fig. 1, le). 2,3 Obviously, a prominent role of the surrounding protein has been anticipated for triggering cobalt-carbon bond activation and enzymatic catalysis. [4][5][6][7][8][9] In this context, most studies have focused so far on the impact of protein interactions with the upper axial ligand of the cofactor, whereas the interactions of the protein from the opposite site are less considered. [10][11][12][13][14] Indeed, B 12 cofactors are anchored to B 12 -dependent enzymes in two different ways: (i) the cofactor retains its intramolecularly coordinating Dmbz base ("base-on" form) during cofactor incorporation into the protein (e.g. class II ribonucleotide reductase or diol dehydratase) or (ii) the intramolecularly coordinating Dmbz base of the cofactor is replaced by a protein histidine ("His") anchoring group ("His-on" conguration). This mode of protein-cofactor binding is shown in Fig. 1 (ref. 10) and encountered, amongst others, in MeCbl-dependent methionine synthase (MetH) as well as AdoCbl dependent methylmalonyl CoA mutase (MCM) and glutamate mutase (GluM) ( Table 1). [15][16][17] Furthermore, the Co-bound His is part of a hydrogenbonded amino acid chain, together with an aspartic acid (Asp) and a variable third residue (lysine for MCM or serine for MetH, Table 1). 11,23 The importance of the cofactor anchoring and the hydrogen bonding network for catalysis has been controversially discussed. 13,23 For example, mutation studies with MetH indicated a dramatic loss of activity while replacing either His or Asp by other amino acids in the hydrogen bonded network. 23 A different study focusing on "His-on" AdoCbl dependent isomerases indicate a structural reorganization of the hydrogen bonding network aer substrate binding. 13,24 This behavior is suggested to stabilize the catalytically active cob(II)alamin intermediates by reducing the basicity of the anchoring His ligand. 13 Such behaviour seems very appealing and could represent a general mode of remote control in reactions depending on "His-on" congured cofactor B 12 . Interestingly, a large number of heme-proteins such as globins, cytochromes c and horse radish peroxidase exhibit also a "His-on" type of cofactor attachment. [25][26][27][28] Partial deprotonation of the proximal His ligand within a lower part hydrogen bonding network enables the heme-dependent enzymes to ne-tune their properties and reactivity ("push effect"). [29][30][31][32][33] This type of redox control has been thoroughly investigated for heme proteins, as the Fe III -13 CN protoporphyrin IX complexes, 34-36 but was so far not studied for related mimics of "His on" Cbl@protein complexes. 12,13,24,[37][38][39][40][41][42][43][44] In this context, only one example reported so far the heterogeneous model of a Cob(III)alamin-protein complex, 14 whereas some fascinating immobilized models of heme-protein complexes have recently attracted considerable attention. [45][46][47][48] Insights into the roles of the "His-on" cofactor attachment and the lower part hydrogen bonding network in B 12 -dependent enzymes are therefore still required. In this context, the development of molecular mimics, 13,40,42 enzyme models 44,49 and new supramolecular biomimetic architectures 14 represent attractive assets.
We envisaged developing a new type of immobilized biomimetic model of the hydrogen bonding network, as found in "His-on" B 12 cofactor complexes. This supramolecular assembly ( Fig. 1 right) consists of three independent subunits: (i) a structurally modied metal cofactor complex, (ii) a carboxylate ion and (iii) a hydrophobic silica C18 solid support. In particular, we envisaged to induce intermolecular hydrogen bonding between the immobilized Co III -coordinated imidazole ligand and the carboxylate anion through hydrophobic interactions on the solid support. 30 With this supramolecular immobilization strategy in mind, we began by developing the most important component of the supramolecular assembly: the molecular B 12 derivative, mimicking the "His-on" Co III -complex. This intramolecular coordination compound consists of three subunits: (i) the tetradentate Co III -corrin complex, (ii) a lower (a-) coordinating imidazole unit and (iii) a trans-located cyanide ligand ( Fig. 1,  right). Such a molecular model allows a thorough study of the imidazole-deprotonation inuence on the redox properties (cyclic voltammetry), on the chromophore (UV-vis) and on the trans-inuence exercised on the upper (b-) axial CN group ( 13 C-NMR). A meticulous structural modeling using QM/MM calculations (ESI, Fig. S1 †) suggested the imidazole backbone derivative of B 12 1-H + as a close biomimetic of the "His-on" conguration in B 12 -cofactor protein complexes (Fig. 2).
. Middle: Cbl bound to the regulatory triad in the "histidine ("His")-on" form, as found in several B 12 -dependent enzymes (X ¼ donor atom, Table 1). Right: The corresponding biomimetic supramolecular model 1 with imidazole hydrogen-bonding interaction (charges are omitted). In this compound, the imidazole subunit and the corrin macrocycle are connected via the f-side chain by an ethylenediamine spacer and a proline subunit. 50-52 1-H + was synthesized as triuoroacetic acid (TFA) salt in three steps with an overall yield of 19% using common peptide coupling chemistry (ESI, Schemes S1 and S2 †). The UV-vis spectrum of 1-H + resembles that of vitamin B 12 (ESI, Fig. S2 †), and the high resolution mass spectrum displays a signal at m/z ¼ 610.30054 (calculated: The structural evaluation of the B 12 @protein model 1-H + was investigated using 2D-ROESY. Through-space correlation was observed between H im 2 (blue on Fig. 3) and the lower side chains 7A and 81 as well as the methyl group 51, all of which are situated at the northern face of the corrin ring. These interactions are complementary to correlations at the southern face of the molecule, between H im 5 and the corrin side chains 151, 131, as well as the 1R situated on the peptide backbone (red on Fig. 3). These data thus unambiguously indicate a coordination of the imidazole group through the N3-nitrogen. These measurements also allowed determining the positioning of the imidazole unit with respect to the macrocycle, and revealed that it follows the C51-C151 axis of the corrin ring (Fig. 3B). Furthermore, comparison with the active site of a "His-on" bound B 12 -cofactor from crystal structure indicates a good agreement with the structural behavior of 1-H + (Fig. 2).
Reversible proton release of 1-H + (Scheme 1) was then tested by pH titration (from pH 8.5 to pH 12.5) and was detected with UV-vis spectroscopy. It revealed a bathochromic shi of 8 nm for both the a and b bands (ESI, Fig. S3 †) which indicates clearly that upon proton release, the imidazole moiety transforms to a stronger s-donating imidazolate ligand. 24,53-55 Additionally, a pK a value of 10.8 was determined for Nd (ESI, Fig. S3 †), which is about 4 pH units lower compared to free imidazole (pK a ¼ 14.5) 56 and comparable to the values observed in His-anchored Fe III -13 CN protoporphyrin IX@cytochrome c complexes (pK a ¼ 10.1 to 10.6). 36 The electrochemical properties of the cobalt center in 1-H + and 1 were examined with cyclic voltammetry. 57-59 At pH 8.5, a cathodic peak at À920 mV vs. Ag/AgCl was observed for 1-H + ( Table 2, entry 2; Fig. S4 †). We assign the cathodic wave to a two electron reduction of the octahedral coordinated Cob(III)alamin in a d 6 -electron conguration to a Co I -square planar complex in a d 8 -electron conguration, in agreement with electrochemical studies with B 12 . 57,60 Switching from compound 1-H + to 1 (pH 12.5) with a Co III -coordinated imidazolate ligand signicantly shied the cathodic peak by 180 mV to a value of À1100 mV vs. Ag/AgCl ( Table 2, entry 1; ESI, Fig. S4 †). Deprotonation of 1-H + to 1 thus stabilizes the Co III form against reduction.
The inuence of the axial ligand basicity on the trans-located b-CN moiety was then investigated with NMR spectroscopy. The measurements were performed at two different pH (pH 7.0 and 12.5), with an isotopically labeled 13 CN ligand on the b-side of the cobalt. The 13 C-NMR of 1-H + revealed a broad signal at  127 ppm ( 13 CN moiety) which shis downeld (133 ppm) upon deprotonation (1) (ESI, Fig. S5 †). This behavior is explained by the coordination of a stronger s-donor (imidazolate, 1), which results in a decreased polarization of the trans-located cyanide p-electron density. 61,62 The broadening of the 13 CN lines of 1 and 1-H + (375 vs. 150 Hz) is due to the distinct nuclear quadrupolar properties of 59 Co, which signicantly increase the t1 and t2 relaxation rates of the directly bound carbon. These effects also prevent the observation of resolved 13 C-multiplets due to scalar coupling between 13 C and 59 Co. Other broadening mechanisms like anisotropic effects or paramagnetic impurities can be excluded, because all remaining 13 C resonances of the measured compounds are sharp (ESI, Fig. S5 †).
Control experiments (CV, UV-vis, 13 C-NMR) were performed with a 'blocked' model at pH 7.0 and pH 12.5 in the same manner described for 1-H + and 1 (ESI, Fig. S6 †). B 12 was used for this purpose, since it contains an a-coordinating Dmbz ligand resembling the imidazole unit, for which reversible protonation of both nitrogens is impossible (Fig. 1, le). In effect, all control experiments with B 12 exhibited no signicant shis upon pH changes, thus proving that all results obtained with 1-H + and 1 originated from the deprotonation of the imidazole ligand.
Plotting the position of the cathodic peak E pc against the upper cyanide 13 C-NMR shi for the 'blocked' model B 12 , the biomimetic compounds 1 and 1-H + as well as two other compounds which present distinct lower axial ligand (aquacyanocobinamide, aquaCNCbi; Fig. 4, right; and dicyanocobinamide, diCNCbi; Fig. 4, right) gives a more extensive picture on the inuence of lower ligand modulation (Fig. 4). 13,24,64,65 The linear correlation suggests that the relative s-donating capacities of the lower ligand similarly inuence both the cathodic peak (E pc ) and the upper CN 13 C shi. Furthermore, by considering that aquaCNCbi represents a model for base-off B 12 , important insights are extracted on how Cbls and Cbl@protein complexes may alter their electrochemical properties in biological systems. Indeed, when considering B 12 alone, the tuning of the redox properties is reached only through the "base-on/base-off" switch, where the a-Dmbz ligand is replaced by the weaker electron donating H 2 O molecule. However, this modication only leads to the destabilization of the cobalt center, as shown by the less negative E pc value for the Co(II)/Co(I) reduction of aquaCNCbi (E pc ¼ À749 mV vs. Ag/AgCl, Table 2, entry 4). The contrary effect, a stabilization of the metal ion seems not to be possible in this coordination mode. Switching the a-Dmbz ligand of B 12 to an imidazole moiety, as encountered in 1-H + , exhibits only a small effect on the Co III /Co I reduction (DV ¼ À23 mV, Table 2, entry 2 and 3). However, deprotonation to an imidazolate moiety (1) is now possible and leads to a substantial cathodic shi of À203 mV ( Table 2, entry 1-3). The lower ligand modulation of 1-H + thus leads to a doubling of the available redox range (Fig. 4, À749 to À1100 mV vs. Ag/AgCl) compared to B 12 alone (Fig. 4, À749 to À897 mV vs. Ag/AgCl). This behavior underscores strikingly that (partial) deprotonation of the coordinated His-residue offers B 12 -dependent proteins an elegant tool for ne-tuning their electron donating properties on demand. The lower ligand modulation is also reected in a bathochromic shi of the aband (Table 2) and the b-band wavelengths (data not shown). Furthermore, linear correlations are observed between the E pc and the a-band (Fig. 4) or the b-band (data not shown). This behavior is in line with recent spectroscopic and theoretical studies, which show that the position of the a-band reects the d z2 contribution of the lower coordinating ligand to the HOMO orbital. 24,[53][54][55] In His-on Cbl complexes, the deprotonation of the His residue is achieved through an H-bonded chain in between the ligand triad. However, under aqueous conditions, deprotonation was only achieved at pH values above the pK a of 1-H + (10.8). For mimicking the biological situation with the model 1-H + more closely, we thus decided to investigate the interactions between the imidazole of 1-H + and an exogenous carboxylate as H-bonding acceptor. For this purpose, we applied rst benzoic acid in dioxane-H 2 O mixtures of 1-H + , 67 which did not result in any spectroscopic changes. Immobilized 1-H + (1-H + SP ) on C18 silica material behaved differently and allowed studying proton release with diffuse reectance spectroscopy, an approach recently introduced by our group. 14 1-H + SP remains base-on upon immobilization, as indicated by the characteristic reectance spectrum of a "base-on" Cbl (ESI, Fig. S7 †). In agreement with studies under homogenous Scheme 1 pH equilibrium between 1-H + and 1. conditions, treatment of 1-H + SP with a strong base (1 M NaOH) led to a bathochromic shi (6 nm for the a-band) and indicated imidazole deprotonation ( Fig. 5; ESI, Fig. S7 †). However, the addition of pure toluene, which lacks any H-bond acceptor, to 1-H + SP did not lead to any changes. In contrast, the further addition of benzoate (tetrabutylammonium salt) to this solvent led to a slight, but characteristic bathochromic shi of the a-band (4 nm) and b-band (2 nm) ( Fig. 5; ESI, Fig. S8 †), which suggests hydrogen-bonding between benzoate and the Nd hydrogen of 1-H + SP , similar to the situation proposed for this class of proteins. 10,13 In summary, we present an unprecedented supramolecular assembly mimicking the regulatory ligand triad of "His-on" metal-cofactor@protein complexes. The data presented herein give evidence that deprotonation of an axially coordinating imidazole to form imidazolate stabilizes signicantly (DV ¼ À203 mV) the Co III form of the corrinoid. However, such behavior cannot be achieved with B 12 in its base-on form.   4 Left: Correlation between E pc (Co III /Co I reduction for all compounds, except aquaCNCbi: Co II /Co I reduction) and the 13 C-NMR shift of CN (left axis, -) and the a-band wavelength (right axis, B); B 12 data correspond to both pH 7.0 and pH 12.5. Right: Structure of aquaCNCbi and diCNCbi (charges are omitted). 66 Modulating the corrinoid redox properties with this elegant trick represents therefore a sophisticated strategy for tailoring the electronic properties of Cbls in biological systems.