Nitrile hydratases (NHase) are non-heme Fe(III)-containing, or noncorrinoid Co(III)-containing, microbial enzymes that catalyze nitrile hydration. 1 The iron form has been studied most extensively. The Fe(III) active site is low spin (S ) 1 / 2 ), and ligated by three cysteinates, two peptide amide nitrogens and either a hydroxide or an NO. [2][3][4] Given the high amount of sequence homology in the active site region, it is likely that the Co-NHase active site is virtually identical to Fe-NHase. 1,5 In one of two recent Fe NHase crystal structures 2,4 two of the metal-bound sulfurs appear to be oxidized, one to a sulfenate ( 114 S cys dO) and the other to a sulfinate ( 112 S cys (dO) 2 ). 4 The sulfenate is not observed by mass spectrometry. 6 Sulfenic acids are usually unstable, 7 and metal-sulfenates are readily oxidized to metalsulfinates. 8,9 A few synthetic NHase models containing oxidized sulfurs have been reported; 10-12 however, none of these incorporate a sulfenate, and only one 12 has an open coordination site.Our group has shown that the spin-state and spectroscopic properties of Fe-NHase can be nicely reproduced by six-coordinate Fe(III) model complexes containing two cis-thiolates and imines. 3,13-15 These models lack oxidized sulfurs, yet their spectroscopic properties are remarkably similar to the enzyme, suggesting that two, of the three, cysteinate NHase sulfurs remain unmodified. To understand how the sulfinate and, possibly, the sulfenate sulfur influences the electronic and reactivity properties of NHase, we have synthesized a series of sulfur-ligated, fivecoordinate Co(III) model complexes containing progressively more oxidized sulfurs.Five-coordinate [Co(III)(S 2 Me2 N 3 (Pr,Pr))] + (1) was synthesized in the same manner as its iron analogue. 15 Complex 1 is intermediate spin (S ) 1) over the temperature range 50-300 K (supplemental Figure S-1), and is reversibly reduced at E 1/2 ) -460 mV vs SCE. The average Co-S distance (2.16(2) Å) in 1 (Figure 1) 16 is shorter than most Co(III) thiolates (average ) 2.24 Å). [17][18][19] Azide and SCN -bind quantitatively to 1 at room temperature trans to one of the thiolate sulfurs. 20 Trigonal bipyramidal 1 (τ ) 0.87) 21 is converted to a more square pyramidal (τ ) 0.48) sulfinate/thiolate-ligated complex, [Co(III)(S Me2 (S O2 )N 3 (Pr,Pr))] + (2; Figure 2), 22 upon stirring in air for 3 days. Only one of the two thiolate sulfurs (S(2)) is oxidized, even upon prolonged stirring. Oxidation of S(2) causes the spinstate to change, from S ) 1 (in 1) to 0 (in 2), and the reduction potential to shift cathodically to E 1/2 ) -380 mV vs SCE. The mean S(2)-O(1,2) distance (1.453(2) Å) in 2 falls in the usual range (1.42-1.48 Å). 17,23,24 The Co-S(2) distance in 2 is indistinguishable from Co-S(1) (Figure 2). Both of the Co-S bonds in 2 are slightly shorter than the Co-S bonds in 1, because (1) Kobayashi, M.; Shimizu, S. Nature Biotechnol. 1998, 16, 733-36. (2) Huang, W.; Jia, J.; Cummings, J.; Nelson, M.; Schneider, G.; Lindqvist, Y. Structure 1997, 5, 691-6...

To determine how a substitutionally inert metal can play a catalytic role in the metalloenzyme nitrile hydratase (NHase), a reactive five-coordinate Co(III) thiolate complex ([Co(III)(S(2)(Me2)N(3)(Pr,Pr))](PF(6)) (1)) that resembles the active site of cobalt containing nitrile hydratase (Co NHase) was prepared. This was screened for reactivity, by using low-temperature electronic absorption spectroscopy, toward a number of biologically relevant "substrates". It was determined 1 will react with azide, thiocyanate, and ammonia, but is unreactive toward nitriles, NO, and butyrate. Substrate-bound 1 has similar spectroscopic and structural properties as [Co(III)(ADIT(2))](PF(6)) (2). Complex 2 is a six-coordinate Co(III) complex containing cis-thiolates and imine nitrogens, and has properties similar to the cobalt center of Co NHase. Substrate binding to 1 is reversible and temperature-dependent, allowing for the determination of the thermodynamic parameters of azide and thiocyanate binding and the rates of ligand dissociation. Azide and thiocyanate bind trans to a thiolate, and with similar entropies and enthalpies (thiocyanate: DeltaH = -7.5 +/- 1.1 kcal/mol, DeltaS = -17.2 +/- 3.2 eu; azide: DeltaH = -6.5 +/- 1.0 kcal/mol, DeltaS = -12.6 +/- 2.4 eu). The rates of azide and thiocyanate displacement from the metal center are also comparable to one another (k(d) = (7.22 +/- 0.04) x 10(-)(1) s(-)(1) for thiocyanate and k(d) = (2.14 +/- 0.50) x 10(-)(2) s(-)(1) for azide), and are considerably faster than one would expect for a low-spin d(6) six-coordinate Co(III) complex. These rates are comparable to those of an analogous Fe(III) complex, demonstrating that Co(III) and Fe(III) react at comparable rates when in this ligand environment. This study therefore indicates that ligand displacement from a low-spin Co(III) center in a ligand environment that resembles NHase is not prohibitively slow so as to disallow catalytic action in nonredox active cobalt metalloenzymes.

We have added cyanide to oxidized 1Fe and 2Fe superoxide reductase (SOR) as a surrogate for the putative ferric-(hydro)peroxo intermediate in the reaction of the enzymes with superoxide, and have used vibrational and ENDOR spectroscopies to study the properties of the active-site paramagnetic iron center. Addition of cyanide changes the active-site iron center in oxidized SOR from rhombic high-spin ferric (S = 5/2) to axial-like low-spin ferric (S = 1/2). Low-temperature resonance Raman and ENDOR data show that the bound cyanide adopts three distinct conformations in Fe(III)-CN SOR. On the basis of 13 CN, C 15 N, and 13 C 15 N isotope shifts of the Fe-CN stretching/Fe-C-N bending modes, resonance Raman studies of 1Fe-SOR indicate one near-linear conformation (Fe-C-N angle ∼175°) and two distinct bent conformations (Fe-C-N angles < 140°). FTIR studies of 1Fe-SOR at ambient temperatures reveals three bound C-N stretching frequencies in the oxidized (ferric) state and one in the reduced (ferrous) state indicating that the conformational heterogeneity in cyanide binding is a characteristic of the ferric state and is not caused by freezing-in of conformational substates at low temperature. 13 C-ENDOR spectra for the 13 CN-bound ferric active sites in both 1Fe-and 2Fe-SORs also show three well-resolved Fe-C-N conformations. Analysis of the 13 C hyperfine tensors for the three substates of the 2Fe-SOR within a simple heuristic model for the Fe-C bonding gives values for the Fe-C-N angles in the three substates of ca. 123° (C3), 133°( C2), taking a reference value from vibrational studies of 175° (C1 species). Resonance Raman and ENDOR studies of SOR variants, in which the conserved glutamate and lysine residues in a flexible loop above the substrate binding pocket have been individually replaced by alanine, indicate that the side chains of these two residues are not involved in direct interaction with bound cyanide. The implications of these results for understanding the mechanism of SOR are discussed.

The syntheses and structures of two analogous five-coordinate mixed nitrogen/thiolate-ligated Co2+ and Fe2+ complexes are described and compared to their previously reported Zn2+ and Ni2+ analogues. The linear, single-chain [S2 R2N3(Pr,Pr)]2- (R = H, Me) ligands examined in this study wrap themselves around metal ions in both a clockwise and counterclockwise manner to afford a racemic mixture of chiral, helical molecules. [FeIIS2N3(Pr,Pr)] (1) crystallizes in the monoclinic space group P21/c with a = 7.853(2) Å, b = 8.667(2) Å, c = 26.079(5) Å, β = 90.37(3)°, V = 1775.0(7) Å3, and Z = 4. [CoIIS2 Me2N3(Pr,Pr)] (2) crystallizes in the monoclinic space group P21/c with a = 9.389(2) Å, b = 19.706(3) Å, c = 12.165(2) Å, β = 103.67(2)°, V = 2186(1) Å3, and Z = 4. Trends in helicity and angular distortions in the Fe2+, Co2+, Ni2+, Zn2+ series which correlate with ionic radius are described. It is suggested that ligand constraints are responsible for the increasing distortion observed (Fe ∼ Zn < Co ≪ Ni) in these structures and that similar constraints may alter the geometries of metalloenzyme active sites.

Thiocyanate hydrolase (TCH) is a pink bacterial metalloenzyme found in Thiobacillus thioparus THI 115 that catalyzes the conversion of thiocyanate to carbonyl sulfide and ammonia. 1,2 Although TCH has not been well characterized spectroscopically, an analysis of the genes encoding for the enzyme has shown a high active-site sequence homology to the metalloenzyme nitrile hydratase (NHase), including the residues that bind the metal center. 2 NHases catalyze the hydration of nitriles to amides and fall into a rare class of cysteine-ligated non-corrinoid Co(III) or non-heme Fe(III) enzymes. 3 NHase has been characterized by a number of physical techniques including EPR, 4 EXAFS, 5,6 Mössbauer, 7 Resonance Raman, 8 and X-ray crystallography. 9,10 The metal in NHase is low-spin and is ligated by three cysteinates, two amide nitrogens (from the peptide backbone), and a hydroxide or water. Given the large amount of sequence homology, it is likely that the metal center of TCH is ligated in a similar fashion.