The Hydrogenobyric Acid Structure Reveals the Corrin Ligand as an Entatic State Module Empowering B12 Cofactors for Catalysis

Abstract The B12 cofactors instill a natural curiosity regarding the primordial selection and evolution of their corrin ligand. Surprisingly, this important natural macrocycle has evaded molecular scrutiny, and its specific role in predisposing the incarcerated cobalt ion for organometallic catalysis has remained obscure. Herein, we report the biosynthesis of the cobalt‐free B12 corrin moiety, hydrogenobyric acid (Hby), a compound crafted through pathway redesign. Detailed insights from single‐crystal X‐ray and solution structures of Hby have revealed a distorted helical cavity, redefining the pattern for binding cobalt ions. Consequently, the corrin ligand coordinates cobalt ions in desymmetrized “entatic” states, thereby promoting the activation of B12‐cofactors for their challenging chemical transitions. The availability of Hby also provides a route to the synthesis of transition metal analogues of B12.

The unique structural [1] and biosynthetic features [2] of coenzyme B 12 and its biological homologues raise fundamental questions concerning the evolution and selection of the corrin ligand, [3] as well as the adoption of B 12 cofactors into key metabolic roles across the three domains of life.T he combined selection of the corrin macrocycle and of cobalt as the specific transition metal center for bio-organometallic catalysis is an intriguing aspect of the B 12 cofactors. [4] The resistance of cobalt corrins against the removal of cobalt without concomitant destruction of the corrin ligand [5] has made astudy of cobalt-free natural corrins amajor scientific challenge. [6] Consequently,d espite the 40 years since vitamin B 12 was prepared by total synthesis, [7] the special partnership of the ligand and the cobalt ion of the natural B 12 cofactors remains largely unexplored. [4a] Tw op athways for B 12 biosynthesis have highlighted intriguing "ring contraction" steps [2] that tailor the "coordination hole" of the tetrapyrrolic macrocycle to the effective size of cobalt ions. [4a,8] Surprisingly,B 12 so wn ligand, hydrogenobyric acid (Hby) ( Figure 1), is not ab iosynthetic intermediate in either of them. [2] However,metabolic engineering of the B 12 biosynthetic pathway has allowed the development of strategies to access metal-free corrins by design. [2b, 9] We recently reported recombinant E. coli strains that generated metal-free corrins,such as hydrogenobyrinic acid a,c-diamide (HBAD). [9,10] Normally,int he aerobic B 12 biosynthetic pathway, HBAD is next chelated with cobalt. [2] However,w hen grown in the absence of cobalt, some purple sulfur bacteria produce cobalt-free corrinoids, [11] including ac ompound tentatively identified as Hby, [11b,c] providing hope for the biological synthesis of Hby.
[2b] Herein, we describe an engineered B 12 biosynthesis pathway variant containing the enzyme CobQ for the effective preparation of Hby,a nd present athorough analysis of the structure of this metal-free corrin, which is critical for binding cobalt ions and for bestowing B 12 biocatalysts with their exceptional reactivity. [4a, 12] Ap athway variant was explored for the biosynthesis of Hby by integrating cobQ from ap urple sulfur bacterium [11a] into the existing repertoire of HBAD biosynthetic genes to generate aH by-operon in an E. coli strain called ED661.
With the Hby-operon integrated in the genome under the control of aT7promoter, Hby was found to be excreted into the culture medium. A4 Lf ermentation of this strain furnished 11.8 mg (12.8 mmol) of crystalline Hby ( Figure 1 and Supporting Information, SI), providing an unprecedented opportunity to study am etal-free natural corrin. When buffered to pH 5-7, and kept in the dark, aqueous solutions of Hby were found to be relatively stable at room temperature (at higher pH Hby was converted into "yellow corrinoids"). [11a,b] In aqueous solution, Hby exhibited UV/Vis absorption [11b] with maxima at 270 nm, 330 nm, 499 nm and 524 nm, and emitted fluorescence with maxima at 552 and 609 nm ( Figure 2), comparable to an atural "metal-free red corrin". [13] Thea bsorption and emission maxima (at 524 and 552 nm, respectively) position the lowest singlet excited state of Hby at 223 kJ mol À1 (for additional data see SI, Figure  Tw ol owfield signals gave evidence for two "inner" H-atoms at N2 and N4, specifying the structure of the cationic corrin ligand core in metal-free Hby.O ther NH tautomers,s uch as 1,3 Hby with "inner" Hatoms at N1 and N3, were not detected ( Figure 2). However,t he HN2 and HN4 protons undergo unsymmetrical transannular H-bonding with N1 and N3, detected with 15 N-labelled Hby,c larifying the question [6] of the location and H-bonding pattern of the "inner" Hatoms in anatural metal-free corrin. TheHatoms H(N2) and H(N4) of Hby were also observed to interact mutually by NOE correlations and by an additional nonbonding through-space interaction, diagnosed through substitution of either one of these H(N)s by D( see SI, Figure S4).
Both of the two "inner" Hatoms are tightly bound by the corrin ligand, despite their fast exchange with water with rates of 21.9 s À1 (HN2) and 6.3 s À1 (HN4) at 308 K(SI, Figure S5). Indeed, the corrin moiety of Hby,aweak acid with pK a -(Hby) = 11.2, [11b] is deprotonated at the corrin periphery, presumably at C8 (Figure 2), as was first deduced by Eschenmoser and Fischli for the model corrin HCor + (formula and crystal structure in SI, Figure S6). [6,14,15] Poignantly,amonoprotonated "neutral" corrin ligand [6] remains elusive.These features of Hby are supported by DFT analyses, which are consistent with the experimentally found stable zwitterionic form of Hby with two unsymmetrical H-bonds N1-HN2 and N3-HN4, support peripheral C8 as the most acidic position of Hby and indicate protomers of Hby with as ingle "inner" Ha tom, either at N4 or at N2, to be significantly less stable (see SI, Figures S7 and S8;T able S5).
Hby generated single crystals from H 2 O/MeCN at 5 8 8C, with space group P2 1 .X -ray analysis revealed ap seudo-C 2symmetric helical arrangement of the core part of Hby,w ith similar structural features observed in the crystal as in solution (Figure 3a nd SI, Figures S6 and S9). Electron density for two "inner" Ha toms was located at N2 and N4, which were at adistance of only 2.27 from each other. The two Ha toms are also close to N1 and N3 with distances of 1.91 and 2.06 ,r espectively,c onsistent with the NMRderived unsymmetrical H-bonding. Thedistance between N2 and N4 of Hby is 3.97 ,that is,about 0.3 longer than that between N1 and N3 (3.67 ). By contrast, in HCor + ,t he "inner" Ha toms are located at N1 and N3. [6,14,15] However, H(N1) of HCor + undergoes H-bonding interactions to an EtOH molecule,g iving the C4-C5 bond of HCor + a2 4.88 8 twist. [6,15] In both, Hby and HCor + ,the "inner" Hatoms break  Figure S1 in the SupportingInformation.  1) at pH 5a nd correlations locating two "inner" HN protons at N2 and N4 and establishing their H-bonds to N1 and N3. Bottom:The structure of Hby in water is represented best by the formula shown, while the tautomer 1,3 Hby (left) was not detected. Deprotonation of Hby generates an iso-corrin anion, presumably iso-Hby À (right), but not a"neutral" corrin (see SI for formulae); R 1 = CH 2 CONH 2 ,R 2 = CH 2 CH 2 CONH 2 in the formulae of 1,3 Hby and iso-Hby À .
the inherent C 2 symmetry of the corrin core,contrasting with the situation in the more regularly structured cobalt corrins and in the "expanded", symmetrical porphyrins. [16] Thec orrin Hby features ac oordination hole with an average diameter of 3.83 ,i ndicating an effective ring contraction of roughly 0.3 ,compared to octaethylporphyrin (HOEP). [16] Hence,t he effective coordination radius in Hby (1.916 )i sc lose to the average equatorial (Co-N) bond in AdoCbl (1.897 ), [17] MeCbl (1.898 ) [18] and in Cbl II (1.88 ). [19] At first sight, the corrin ligand appears to be well adapted for coordination of Co III and Co II ions. [17][18][19] However,t he corrin-specific trans junction between rings A and Di mposes ad istinctly helical structure. [1] Consequently, the four chelating Na toms of the corrin macrocycle of Hby represent as crew-like coordination hole,l eading to ac oordinative misfit for cobalt ions that is particularly strong for Co III .
Them utual conformational adaptation of the corrin ligand and the coordinated cobalt ions was evaluated by two structure parameters:i )The corrin helicity h of the innermost coordination space of the corrin ligand provided by the four corrin nitrogen atoms,defined by the dihedral angle N1-N2-N3-N4 (see Figure 4). In the metal-free corrin Hby it amounts to h(Hby) = 12.98 8.C o III corrins feature strongly reduced h values,e.g., h(AdoCbl) = 3.58 8 and h(MeCbl) = 4.68 8. Hence,t he ligand is strongly flattened by Co III binding in AdoCbl and MeCbl.O nt he other hand, the four-coordinate Co II center (Cbl II ACA) of the human adenosyl-transferase ACAf its the corrin ligand better, displaying h(Cbl II ACA) = 88 8. [20] Five-coordinate Co II corrins display lower intermediate levels (see Figure 4). ii)The interplanar angle f,w hich concerns the equatorial coordination sphere at the cobalt center, indicating coordinative strain in cobalt corrins when deviating from 08 8 (see Figure 4a nd SI for details). The reference value of Hby is f = 13.58 8. In Cbl II ACA f = 178 8,i n the two Co II corrins, Cbl II and Cbin II [21] f is 12.58 8,respectively 7.68 8.InCo III corrins,like AdoCbl and MeCbl, f is only 4-58 8.
Hence, h and f decrease in aroughly correlated fashion from Hby to Co II and to Co III corrins,i ndicating significant directional coordinative misfit in Co III corrins.
Thestructural analysis of the helical corrin ligand Hby of B 12 derivatives has revealed key elements helping to "demystify vitamin B 12 ". [3,4] It has confirmed the postulated "fit" [3,4,8] of the "ring-contracted" corrin ligand Hby to the size of Co III and Co II ions (in AdoCbl and Cbl II ). However,t he corrin ligand Hby is distinctly helical, dissatisfying the octahedral coordination preference of Co III centers,while better meeting the requirements of Co II and Co I ions (Figures 4a nd 5). The inferior accommodation of Co III over Co II centers implies ap reviously overlooked coordinative strain for Co III corrins that promotes homolytic (Co-C) bond cleavage.This effect is crucial for the homolysis of AdoCbl to Cbl II in the B 12dependent radical isomerization reactions. [4c, 23] Thesame type of strain also activates the cobalt-bound methyl group of Figure 3. The ring-contracted corrin ligand is auniquely skewed helix. Topleft:T wo projections of the crystal structure of Hby (color coding: carbons of corrin core:r ed;ofsubstituents:green;nitrogens:blue; oxygens:red;hydrogens:white). Topcenter and right:P rojectionso f core structures of Hby (red), of octaethyl-porphyrin (HOEP,black) and their superposition (middle).B ottom:C ore structures of the metal-free corrins Hby and HCor + , [14,15] and of the Cbls Cbl II and AdoCbl,i nwhich effects of "inner" Hatoms or of Co ions on the lengths of diagonals are highlighted. The interplanar angle f is large in helical Hby and in Cbl II ,but strongly reduced in AdoCbl.Infour-coordinate Co II and six-coordinate Co III porphyrins (CoOEP) f = ca. 08 8. [16] c) Cylinder projections of the structures of Hby, AdoCbl and Cbin II ,highlighting conformational differences in the corrin ligand. The conformationo f Hby (black trace) is largely retained in the Co II corrin Cbin II (blue trace), contrastingw ith its stronger adaptation to Co III binding in AdoCbl (red trace).
MeCbl for abstraction by radicals [24] in B 12 -dependent radical SAM enzymes. [25] As imilar strain decrease may also accompany the heterolytic abstraction of the cobalt-bound methyl of MeCbl by nucleophiles in B 12 -dependent enzymatic methyl group transfer,p roducing Co I cobalamin. [26] In the critical adenosyl-transferase ACA, an unstable four-coordinate form of Cbl II (Cbl II ACA) [20] undergoes the reduction to the fourcoordinate Co I species.S uch essential four-coordinate Co II and Co I forms,which are hard to generate metabolically, [25b,27] appear to be well accommodated by the helical coordination hole of the corrin ligand. Since Co I corrins are not structurally characterized, model DFT calculations were used. They indicate ar eduction of coordinative strain, by about 7kJmol À1 ,f or the transition from six-coordinate Co III to four-coordinate Co I ions,w hen bound by four Na toms in anonplanar arrangement, as in Hby.T he analogous Co III -to-Co II transition experiences as train decrease of about 10 kJ mol À1 (SI, Figure S10). Hence,t he inherently helical corrin ligand acts as a" Procrustean bed" that destabilizes Co III centers towards loss of axial ligands and formation of Co II or Co I forms,e nhancing catalysis by the B 12 cofactors.
Thep reviously unrecognized role of the flexible helical corrin ligand in activating organometallic Co III corrins for catalysis classifies the B 12 cofactors AdoCbl and MeCbl as "entatic state" molecules.T he term "entatic" state was initially applied to proteins with metal centers bound in astrained coordination sphere to lower activation barriers for enzyme catalysis. [28] Herein, we infer that cobalt corrins have been selected [4a] since they represent "entatic state" complexes in which ligand-imposed strain activates Co III centers for catalysis.Arelated situation exists in coenzyme F 430 ,aNi corphinoid, in which radial strain results from am isfit between the size of the coordinated Ni ions and the porphyrinoid macrocycle. [4a,29] Theavailability of the metal-free Hby has also opened the door to the direct preparation of transition metal analogues of the cobalamins,the "metbalamins" (Metbls), a" Holy Grail" of bioinorganic chemistry. [6, 9, 11b,c,30] Hence, Hby has served as an effective starting material for the synthesis of transition metal B 12 analogues,tobe reported shortly.Asdescribed with AdoRhbl,the Rh III analogue of AdoCbl, [9] suitably structured Metblsh old as ignificant potential as "antivitamins B 12 ", in biological imaging or as novel antibiotics. [31] Thee xciting prospect of investigations with transition metal complexes of the skewed corrins will interest experimental scientists and theoretical chemists alike.

Experimental Section
CCDC 1881269 (Hby,s ee SI) contains the supplementary crystallographic data for this paper.These data can be obtained free of charge from TheCambridgeC rystallographic Data Centre. Figure 5. The helical corrin ligand binds cobalt centers in astrained state, promotingthe cleavage of axial bonds and formation of reduced corrinoids. This is symbolized at the top for the Co III corrin AdoCbl (before and after Co-C bond cleavage), for four-coordinate Co II and Co I cobalamin, and for Hby.M iddle and bottom:The corrin ligand is flattened and interplanar angle f decreased most strongly at Co III centers, less at Co II and Co I ions. Both parameters indicate an increasing misfit and strain in the series Co I /Co II and Co III corrins; numericald ata for h and f are collected in Figure 4.