Proton transfer in carbonic anhydrase is controlled by electrostatics rather than the orientation of the acceptor

Combined quantum mechanical/molecular mechanical (QM/MM) simulations are carried out to analyze factors that dictate the proton transfer in carbonic anhydrase II (CAII), an enzyme that has been used as a prototypical example of long-range proton transfers in biomolecules. In contrast to the long-held conjecture in the experimental literature, the computed potentials of mean force (PMF) suggest that the proton transfer in CAII is not very sensitive to the orientation of the acceptor group (His 64) and, therefore, the number of water molecules that bridge the donor (zinc-water) and acceptor groups. Perturbative analysis indicates that a series of polar and charged residues close to the transfer pathways make the dominant contribution to the barrier and exothermicity of the proton transfer reaction, thus supporting the proposal from previous studies of Warshel and co-workers using a somewhat simpler QM/MM model that electrostatic interactions play a major role in the proton transfer in CAII. The PMF results are in striking contrast to previous analysis using the same QM/MM method but an ensemble of minimum energy path (MEP) calculations, which found a steep dependence of the barrier height on the number of bridging water molecules. Analysis of the configurations sampled in the PMF and MEP simulations suggests that this difference arises because the PMF simulations sample a largely stepwise mechanism while the local MEP calculations artificially favored concerted transfers due to the specific protocol used to generate the initial configurations. Therefore, this study presents a compelling argument for carrying out proper conformational sampling in the study of long-range proton transfers. Finally, we illustrate that Phi analysis, which has been widely used in protein folding studies, can potentially generate new mechanistic information for long-range proton transfers regarding the sequence of events. The results of the perturbation analysis and the Phi analysis provide opportunities for experimentally testing the mechanistic proposals from this study and our recent work in which a stepwise "proton hole" transfer pathway has been proposed.     

Polymerase-Tailored Variations in the Water-Mediated and Substrate-Assisted Mechanism for Nucleotidyl Transfer: Insights from a Study of T7 DNA Polymerase

The nucleotidyl transfer reaction catalyzed by DNA polymerases is the critical step governing the accurate transfer of genetic information during DNA replication, and its malfunctioning can cause mutations leading to human diseases, including cancer. Here, utilizing ab initio quantum mechanical/molecular mechanical calculations with free-energy perturbation, we carried out an extensive investigation of the nucleotidyl transfer reaction mechanism in the well-characterized high-fidelity replicative DNA polymerase from phage T7. Our defined mechanism entails an initial concerted deprotonation of a conserved crystal water molecule with protonation of the γ-phosphate of the deoxynucleotide triphosphate(dNTP) via a solvent water molecule, and then the proton on the primer 3′-terminus is transferred to the resulting hydroxide ion. Subsequently, the nucleophilic attack takes place, with the formation of a metastable pentacovalent phosphorane intermediate. Finally, the pyrophosphate leaves, facilitated by the relay of the proton on the γ-phosphate to the α-β bridging oxygen via solvent water. The computed activation free-energy barrier is consistent with kinetic data for the chemistry step with correct nucleotide incorporation in T7 DNA polymerase. This variant of the water-mediated and substrate-assisted mechanism has features tailored to the structure of the T7 DNA polymerase. However, a unifying theme in the water-mediated and substrate-assisted mechanism is the cycling through crystal and solvent water molecules of the proton originating from the primer 3′-terminus to the α-β bridging oxygen of the deoxynucleotide triphosphate; this neutralizes the evolving negative charge as pyrophosphate leaves and restores the polymerase to its pre-chemistry state. These unifying features are likely requisite elements for nucleotidyl transfer reactions.     

A quantum mechanics/molecular mechanics study of the catalytic mechanism of the thymidylate synthase

A theoretical study of the molecular mechanism of the thymidylate synthase-catalyzed reaction has been carried out using hybrid quantum mechanics/molecular mechanics methods. We have examined all of the stationary points (reactants, intermediates, transition structures, and products) on the multidimensional potential energy surfaces for the multistep enzymatic process. The characterization of these relevant structures facilitates the gaining of insight into the role of the different residues in the active site. Furthermore, analysis of the full energy profile has revealed that the step corresponding to the reduction of the exocyclic methylene intermediate by hydride transfer from the 6S position of 5,6,7,8-tetrahydrofolate (H4folate), forming dTMP and 7,8-dihydrofolate (H2folate), is the rate-limiting step, in accordance with the experimental data. In this step, the hydride transfer and the scission of an overall conserved active site cysteine residue (Cys146 in Escherichia coli) take place in a concerted but very asynchronous way. These findings have also been tested with primary and secondary deuterium, tritium, and sulfur kinetic isotope effects, and the calculations have been compared to experimental data. Finally, the incorporation of high-level quantum mechanical corrections to the semiempirical AM1 Hamiltonian into our hybrid scheme has allowed us to obtain reasonable values of the energy barrier for the rate-limiting step. The resulting picture of the complete multistep enzyme mechanism that is obtained reveals several new features of substantial mechanistic interest.     

Quantum mechanical study of antioxidative ability and antioxidative mechanism of rutin (vitamin P) in solution

A quantum mechanical approach has been used to shed light on the antioxidative mechanism for scavenging hydroxyl radicals (OH) and superoxide radicals () by rutin in the solution phase. Density-functional theory (DFT) using B₃LYP and UB₃LYP functional and split-valance 6-311+G^∗∗ basis sets were used to optimize rutin and its different radical forms. Analysis of the theoretical bond dissociation enthalpy (BDE) values for all OH sites of rutin in solution clearly shows the importance of the B-ring and the 3′-OH and 4′-OH groups in the antioxidant activity. We have also investigated the spin density of the radicals to determine the delocalization possibilities. The results of the calculations showed that the oxidation of rutin by both the hydroxyl radical and superoxide radical is an exothermic reaction. In all calculations solvent effects were considered using a polarized continuum model (PCM).     

Properties of intramolecular proton transfer in carbonic anhydrase III.

We investigated the efficiency of glutamic acid 64 and aspartic acid 64 as proton donors to the zinc-bound hydroxide in a series of site-specific mutants of human carbonic anhydrase III (HCA III). Rate constants for this intramolecular proton transfer, a step in the catalyzed dehydration of bicarbonate, were determined from the proton-transfer-dependent rates of release of H2 18O from the enzyme measured by mass spectrometry. The free energy plots representing these rate constants could be fit by the Marcus rate theory, resulting in an intrinsic barrier for the proton transfer of deltaG0++ = 2.2 +/- 0.5 kcal/mol, and a work function or thermodynamic contribution to the free energy of reaction wr = 10.8 +/- 0.1 kcal/mol. These values are very similar in magnitude to the Marcus parameters describing intramolecular proton transfer from His64 and His67 to the zinc-bound hydroxide in mutants of HCA III. That result and the equivalent efficiency of Glu64 and Asp64 as proton donors in the catalysis by CA III demonstrate a lack of specificity in proton transfer from these sites, which is indirect evidence of a number of proton conduction pathways through different structures of intervening water chains. The dominance of the thermodynamic contribution or work function for all of these proton transfers is consistent with the view that formation and breaking of hydrogen bonds in such water chains is a limiting factor for proton translocation.     

DFT study of the gas phase proton transfer in guanine assisted by water, methanol, and hydrogen peroxide

A computational study of hydrogen-bonded complexes between the oxo-/hydroxo-amino N7/9H tautomers of guanine and water, methanol, and hydrogen peroxide has been performed at the B3LYP/6-31+G(d) level of theory. The mechanisms of the water-, methanol-, and hydrogen peroxide-assisted proton transfers in guanine were studied and compared with the intramolecular proton transfer in guanine in the gas phase. It was found that the assisted proton transfers pass through about three times lower energy barriers than those found for isolated guanine tautomers. Figure DFT study of the gas phase proton transfer in guanine assisted by water, methanol and hydrogen peroxide     

Computational and experimental studies on the catalytic mechanism of biliverdin-IXbeta reductase

BVR-B (biliverdin-IXbeta reductase) also known as FR (flavin reductase) is a promiscuous enzyme catalysing the pyridine-nucleotide-dependent reduction of a variety of flavins, biliverdins, PQQ (pyrroloquinoline quinone) and ferric ion. Mechanistically it is a good model for BVR-A (biliverdin-IXalpha reductase), a potential pharmacological target for neonatal jaundice and also a potential target for adjunct therapy to maintain protective levels of biliverdin-IXalpha during organ transplantation. In a commentary on the structure of BVR-B it was noted that one outstanding issue remained: whether the mechanism was a concerted hydride transfer followed by protonation of a pyrrolic anion or protonation of the pyrrole followed by hydride transfer. In the present study we have attempted to address this question using QM/MM (quantum mechanics/molecular mechanics) calculations. QM/MM potential energy surfaces show that the lowest energy pathway proceeds with a positively charged pyrrole intermediate via two transition states. These initial calculations were performed with His(153) as the source of the proton. However site-directed mutagenesis studies with both the H153A and the H153N mutant reveal that His(153) is not required for catalytic activity. We have repeated the calculation with a solvent hydroxonium donor and obtain a similar energy landscape indicating that protonation of the pyrrole is the most likely first step followed by hydride transfer and that the required proton may come from bulk solvent. The implications of the present study for the design of inhibitors of BVR-A are discussed.     

Theoretical investigation of the dissociative interchange (Id) mechanism for water exchange on magnesium(II) in aqueous solution

A dissociative (D) and a solvent-assisted dissociative interchange (I_d) water-exchange pathways for magnesium(II) in aqueous solution were simulated with density functional theory calculations. The D mechanism of includes a five-coordinated intermediate, while the I_d water-exchange pathway of proceeds with the assistance of a solvent water molecule, which supports the experimental assignment of the reaction mechanism. The intrinsic activation volume was used to differentiate between I_d and I_a mechanisms despite of the exclusion of the contribution of transmission coefficient. The calculated intrinsic activation volume for the I_d mechanism is consistent with the experimental data, and is closer to the experimental data than that for D mechanism. The I_d mechanism is suggested as the dominate water-exchange pathway of depending on the intrinsic activation volume with the assistance of the activation entropy. The calculations also showed that the influences of the explicit and bulk waters on energy barriers for D and I_d mechanisms are obviously different.     

Ground state intermolecular proton transfer in the supersystems thymine–(H2O)n and thymine–(CH3OH)n, n = 1,2: a theoretical study

Twelve binary and eight ternary supersystems between thymine and methanol, and water were investigated in the ground state at the B3LYP and MP2 levels of theory using B3LYP/6-311 + + G(d,p) basis functions. The thermodynamics of complex formations and the mechanisms of intermolecular proton transfers were clarified in order to find out the most stable H-boned system. It was established that the energy barriers of the water/methanol-assisted proton transfers are several times lower than those of the intramolecular proton transfers in the DNA/RNA bases. The X-ray powder spectra of thymine, and this precrystallized from water and methanol showed that water molecules are incorporated in the crystal lattice of thymine forming H-bridges between thymine molecules. Figure Intermolecular H-bonding of thymine     

Catalytic mechanism of aldose reductase studied by the combined potentials of quantum mechanics and molecular mechanics

The catalytic reduction of

Full-size image

-glyceraldehyde to glycerol by aldose reductase has been investigated with the combined potentials of quantum mechanics (QM) and molecular mechanics (MM) to resolve the question of whether Tyr48 or His110 serves as the proton donor during catalysis. Site directed mutagenesis studies favor Tyr48 as the proton donor while the presence of a water channel linking the Nδ1 of His110 to the bulk solvent suggests that His110 is the proton donor. Utilizing the combined potentials of QM and MM, the binding mode of substrate

Full-size image

-glyceraldehyde was investigated by optimizing the local geometry of Asp43, Lys77, Tyr48, His110 and NADPH at the active site of aldose reductase. Reaction pathways for the reduction of

Full-size image

-glyceraldehyde to glycerol were then constructed by treating both Tyr48 and His110 as proton donors. Comparison of energetics obtained from the reaction pathways suggests His110 to be the proton donor. Based on these findings, a reduction mechanism of

Full-size image

-glyceraldehyde to glycerol is described.     

Kinetics of proton-linked flavin conformational changes in p-hydroxybenzoate hydroxylase

p-Hydroxybenzoate hydroxylase (PHBH) is an FAD-dependent monooxygenase that catalyzes the hydroxylation of p-hydroxybenzoate (pOHB) to 3,4-dihydroxybenzoate in an NADPH-dependent reaction. Two structural features are coupled to control the reactivity of PHBH with NADPH: a proton-transfer network that allows protons to be passed between the sequestered active site and solvent and a flavin that adopts two positions: "in", where the flavin is near pOHB, and "out", where the flavin is near NADPH. PHBH uses the proton-transfer network to test for the presence of a suitable aromatic substrate before allowing the flavin to adopt the NADPH-accessible conformation. In this work, kinetic analysis of the His72Asn mutant, with a disrupted proton-transfer network, showed that flavin movement could occur in the presence or absence of NADPH but that NADPH stimulated movement to the reactive conformation required for hydride transfer. Substrate and solvent isotope effects on the transient kinetics of reduction of the His72Asn mutant showed that proton transfer was linked to flavin movement and that the conformational change occurred in a step separate from that of hydride transfer. Proton transfers during the reductive half-reaction were observed directly in the wild-type enzyme by performing experiments in the presence of a fluorescent pH-indicator dye in unbuffered solutions. NADPH binding caused rapid proton release from the enzyme, followed by proton uptake after flavin reduction. Solvent and substrate kinetic isotope effects showed that proton-coupled flavin movement and reduction also occurred in different steps in wild-type PHBH. These results allow a detailed kinetic scheme to be proposed for the reductive half-reaction of the wild-type enzyme. Three kinetic models considered for substrate-induced isomerization are analyzed in the Appendix.     

Mechanism of proton transfer in the 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni

3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni catalyzes the oxidation of androsterone with NAD(+) to form androstanedione and NADH with a concomitant releasing of protons to bulk solvent. To probe the proton transfer during the enzyme reaction, we used mutagenesis, chemical rescue, and kinetic isotope effects to investigate the release of protons. The kinetic isotope effects of (D)V and (D(2)O)V for wild-type enzyme are 1 and 2.1 at pL 10.4 (where L represents H, (2)H), respectively, and suggest a rate-limiting step in the intramolecular proton transfer. Substitution of alanine for Lys(159) changes the rate-limiting step to the hydride transfer, evidenced by an equal deuterium isotope effect of 1.8 on V(max) and V/K(androsterone) and no solvent kinetic isotope effect at saturating 3-(cyclohexylamino)propanesulfonic acid (CAPS). However, a value of 4.4 on V(max) is observed at 10 mm CAPS at pL 10.4, indicating a rate-limiting proton transfer. The rate of the proton transfer is blocked in the K159A and K159M mutants but can be rescued using exogenous proton acceptors, such as buffers, small primary amines, and azide. The Br?nsted relationship between the log(V/K(d)(-base)Et) of the external amine (corrected for molecular size effects) and pK(a) is linear for the K159A mutant-catalyzed reaction at pH 10.4 (beta = 0.85 +/- 0.09) at 5 mm CAPS. These results show that proton transfer to the external base with a late transition state occurred in a rate-limiting step. Furthermore, a proton inventory on V/Et is bowl-shaped for both the wild-type and K159A mutant enzymes and indicates a two-proton transfer in the transition state from Tyr(155) to Lys(159) via 2'-OH of ribose.     

Density-functional analysis of the electronic structure of tris-bipyridyl Ru(II) sensitisers

Excited state transitions and energies of a series of [Ru(bpy)₃]²⁺ type complexes incorporating the ligand, 4,4′-bis-phosphonato(methyl)-2,2′-bipyridine (dmpbpy) was investigated, and the influence of this organometallic ligand on the electronic structure of the complexes was examined using Time-Dependent Density Functional Theory (TD-DFT). Experimental data and the theoretical TD-DFT calculations were presented to support the effect of non-equivalent ligand substitution on spectral and molecular orbital (MO) energy properties on this class of tris-chelate surface sensitisers. For the series of complexes studied, it was identified that the lowest lying LUMO states were consistently found to reside on the ligand 2,2′-bipyridine (bpy) for gas phase calculations. As an implication of this, it was suggested that this could impact the effectiveness of these complexes as surface sensitisers in PEC cell applications such as the dye-sensitised solar cell (DSC) due to the lower probability of the excited state electron residing on a ligand anchored to the semiconductor substrate. However, further calculations in a solvation medium showed that the electron withdrawing nature of PO₃H₂ on dmpbpy saw the lowest lying LUMO states are populated on dmpbpy. This inhomogeneous distribution of electron density across non-equivalent ligands may have implications for further ‘spectral tuning’ of surface sensitisers. Despite the TD-DFT gas phase calculations not being corrected for solvent/media effects, the three longest wavelength bands associated with known charge transfer phenomena were identified. The symmetry allowed _MLCT in the visible region was assigned as a  ← dπ transition, the mid-UV spectrum _LC was assigned  ← π in origin. Whilst the near-UV shoulder on the blue side of _MLCT showed dσ ← dπ and π∗ ← dπ transitional character and was tentatively described as _MC/MLCT. UV-Vis absorption spectra calculated for solvated analogues containing dmpbpy indicated that the low energy transitions associated with the MLCT are subject to bathochromic shift due to solvent polarity by 0.062 eV (500 cm⁻¹) compared with the gas phase calculations, which is more highly correlated to the observed experimental transitions.     

The proton pumping stoichiometry of purified mitochondrial complex I reconstituted into proteoliposomes

NADH:ubiquinone oxidoreductase (complex I) is the largest and most complicated enzyme of aerobic electron transfer. The mechanism how it uses redox energy to pump protons across the bioenergetic membrane is still not understood. Here we determined the pumping stoichiometry of mitochondrial complex I from the strictly aerobic yeast Yarrowia lipolytica. With intact mitochondria, the measured value of indicated that four protons are pumped per NADH oxidized. For purified complex I reconstituted into proteoliposomes we measured a very similar pumping stoichiometry of . This is the first demonstration that the proton pump of complex I stayed fully functional after purification of the enzyme.     

A proposed proton shuttle mechanism for saccharopine dehydrogenase from Saccharomyces cerevisiae

Saccharopine dehydrogenase [N6-(glutaryl-2)-L-lysine:NAD oxidoreductase (L-lysine forming)] catalyzes the final step in the alpha-aminoadipate pathway for lysine biosynthesis. It catalyzes the reversible pyridine nucleotide-dependent oxidative deamination of saccharopine to generate alpha-Kg and lysine using NAD+ as an oxidizing agent. The proton shuttle chemical mechanism is proposed on the basis of the pH dependence of kinetic parameters, dissociation constants for competitive inhibitors, and isotope effects. In the direction of lysine formation, once NAD+ and saccharopine bind, a group with a pKa of 6.2 accepts a proton from the secondary amine of saccharopine as it is oxidized. This protonated general base then does not participate in the reaction again until lysine is formed at the completion of the reaction. A general base with a pKa of 7.2 accepts a proton from H2O as it attacks the Schiff base carbon of saccharopine to form the carbinolamine intermediate. The same residue then serves as a general acid and donates a proton to the carbinolamine nitrogen to give the protonated carbinolamine. Collapse of the carbinolamine is then facilitated by the same group accepting a proton from the carbinolamine hydroxyl to generate alpha-Kg and lysine. The amine nitrogen is then protonated by the group that originally accepted a proton from the secondary amine of saccharopine, and products are released. In the reverse reaction direction, finite primary deuterium kinetic isotope effects were observed for all parameters with the exception of V2/K(NADH), consistent with a steady-state random mechanism and indicative of a contribution from hydride transfer to rate limitation. The pH dependence, as determined from the primary isotope effect on DV2 and D(V2/K(Lys)), suggests that a step other than hydride transfer becomes rate-limiting as the pH is increased. This step is likely protonation/deprotonation of the carbinolamine nitrogen formed as an intermediate in imine hydrolysis. The observed solvent isotope effect indicates that proton transfer also contributes to rate limitation. A concerted proton and hydride transfer is suggested by multiple substrate/solvent isotope effects, as well as a proton transfer in another step, likely hydrolysis of the carbinolamine. In agreement, dome-shaped proton inventories are observed for V2 and V2/K(Lys), suggesting that proton transfer exists in at least two sequential transition states.     

Importance of a serine proximal to the C(4a) and N(5) flavin atoms for hydride transfer in choline oxidase

Choline oxidase catalyzes the flavin-dependent, two-step oxidation of choline to glycine betaine with the formation of an aldehyde intermediate. In the first oxidation reaction, the alcohol substrate is initially activated to its alkoxide via proton abstraction. The substrate is oxidized via transfer of a hydride from the alkoxide α-carbon to the N(5) atom of the enzyme-bound flavin. In the wild-type enzyme, proton and hydride transfers are mechanistically and kinetically uncoupled. In this study, we have mutagenized an active site serine proximal to the C(4a) and N(5) atoms of the flavin and investigated the reactions of proton and hydride transfers by using substrate and solvent kinetic isotope effects. Replacement of Ser101 with threonine, alanine, cysteine, or valine resulted in biphasic traces in anaerobic reductions of the flavin with choline investigated in a stopped-flow spectrophotometer. Kinetic isotope effects established that the kinetic phases correspond to the proton and hydride transfer reactions catalyzed by the enzyme. Upon removal of Ser101, there is an at least 15-fold decrease in the rate constants for proton abstraction, irrespective of whether threonine, alanine, valine, or cysteine is present in the mutant enzyme. A logarithmic decrease spanning 4 orders of magnitude is seen in the rate constants for hydride transfer with increasing hydrophobicity of the side chain at position 101. This study shows that the hydrophilic character of a serine residue proximal to the C(4a) and N(5) flavin atoms is important for efficient hydride transfer.     

On the signaling mechanism and the absence of photoreversibility in the AppA BLUF domain

The flavoprotein AppA from Rhodobacter sphaeroides contains an N-terminal, FAD-binding BLUF photoreceptor domain. Upon illumination, the AppA BLUF domain forms a signaling state that is characterized by red-shifted absorbance by 10 nm, a state known as AppA_RED. We have applied ultrafast spectroscopy on the photoaccumulated AppA_RED state to investigate the photoreversible properties of the AppA BLUF domain. On light absorption by AppA_RED, the FAD singlet excited state decays monoexponentially in 7 ps to form the neutral semiquinone radical FADH^•, which subsequently decays to the original AppA_RED molecular ground state in 60 ps. Thus, is deactivated rapidly via electron and proton transfer, probably from the conserved tyrosine Tyr-21 to FAD, followed by radical-pair recombination. We conclude that, in contrast to many other photoreceptors, the AppA BLUF domain is not photoreversible and does not enter alternative reaction pathways upon absorption of a second photon. To explain these properties, we propose that a molecular configuration is formed upon excitation of AppA_RED that corresponds to a forward reaction intermediate previously identified for the dark-state BLUF photoreaction. Upon excitation of AppA_RED, the BLUF domain therefore enters its forward reaction coordinate, readily re-forming the AppA_RED ground state and suppressing reverse or side reactions. The monoexponential decay of FAD* indicates that the FAD-binding pocket in AppA_RED is significantly more rigid than in dark-state AppA. Steady-state fluorescence experiments on wild-type, W104F, and W64F mutant BLUF domains show tryptophan fluorescence maxima that correspond with a buried conformation of Trp-104 in dark and light states. We conclude that Trp-104 does not become exposed to solvent during the BLUF photocycle.     

Theoretical studies on the tautomerism of tetrazole selenone

The tautomerism of all possible forms of tetrazole selenone (A–G), induced by proton transfer, was studied, theoretically, in different environments including gas phase, continuum solvent and microsolvated environment with one or two explicit water or ammonia molecules. The calculations were performed using two different levels of theory including mPW2PLYP and DFT-B3LYP. The 6-311++G(d,p) basis set was used for C, H, O and N and the standard relativistic effective core pseudo potential LANL2DZ basis set was used for Se atom. It was found that the tetrazole selenone, in the form of A, is the most stable isomer in all of the environments considered in this work. The kinetics of proton transfer reaction was studied in both gas and solvent environments and it was concluded that the activation energy of the reaction increases with going from the gas phase to polar solvents. Moreover, the proton transfer reaction assisted by one or two water or ammonia molecules was investigated and it was found that the activation energy significantly reduces.     

Mononuclear cobalt(II) carboxylate complexes: Synthesis, molecular structure and selective oxygenation study

Some cobalt carboxylate (both mononuclear as well as binuclear) complexes have been prepared by using hindered hydrotris(3,5-diisopropyl-1-pyrazolyl)borate (Tp^iPr2) as supporting ligand. The reaction of [Tp^iPr2Co(NO₃)] (2) with sodium benzoate resulted in the formation of acetonitrile coordinated complex [Tp^iPr2Co(OBz)(CH₃CN)] (3) whereas the reaction of 2 with sodium fluorobenzoate gave coordinately unsaturated five coordinate complex of the type [Tp^iPr2Co(F-OBz)] (4). The oxidation of compound 4 in the presence of 3,5-diisopropylpyrazole resulted in the formation of a unique compound (5) where only one methine carbon of isopropyl group on pyrazole ring of hydrotris(3,5-diisopropyl-1-pyrazolyl)borate oxidized and coordinated with cobalt center. In compound 5, the binding behavior of fluorobenzoate also changes from bidentate to monodentate and the nonbonded oxygen atom formed intramolecular hydrogen bond with the hydrogen atom of the NH fragment of the coordinated . X-ray crystallography and IR studies confirmed the existence of hydrogen bonding in complex 5. The pyrazolato bridged binuclear cobalt(II) complex (6) was prepared by the reaction of hydrated cobalt(II) nitrate, 3,5-diisopropylpyrazole and sodium nitrobenzoate where, each cobalt is four coordinate. The X-ray structure of 6 showed that the NH fragment of terminally coordinated formed intramolecular hydrogen bonding with nonbonded oxygen atom of monodentately coordinated nitrobenzoate.