The aryne cobalt complex [Co(4-CF 3 -η 2 -C 6 F 3 )(PMe 3 ) 3 ] (1) was formed from the reaction of [Co(PMe 3 ) 4 ] and perfluorinated toluene through selective activation of two C-F bonds of perfluorotoluene. A mechanism for the formation of complex 1 is proposed and in most parts experimentally verified. Following this mechanism, a synergistic effect of an electron-rich cobalt(0) center and one of its trimethylphosphine ligands is responsible for the C-F activation of two carbon-fluorine bonds of perfluorotoluene. The detection of difluorotrimethylphoshphorane as the sole byproduct provides strong evidence for this mechanism. Complex [Co(4-CF 3 -C 6 F 4 )(PMe 3 ) 3 ] (4), an intermediate of the proposed mechanism to the aryne complex, was also isolated and structurally characterized. Complex 4 transforms to complex 1 via activation of a second C-F bond of a perfluorotolyl ligand only in the presence of trimethylphosphine in the reaction mixture. Complex 4 reacts with CO under atmospheric pressure and room temperature to give [Co(4-CF 3 -C 6 F 4 )(CO) 2 (PMe 3 ) 2 ] (6) and with bromobenzene via one-electron oxidative addition of the C-Br bond to give the cobalt(II) bromide [CoBr(4-CF 3 -C 6 F 4 )(PMe 3 ) 3 ] (8) and a C-C-coupling product, 4-phenylheptafluorotoluene (7). The structures of complexes 1, 4, and 8 were determined by X-ray crystallography.
Gaseous molecules are essential for biological processes, yet mapping the migration of gas molecules into and out of proteins represents a significant challenge. Cytochrome P450 enzymes (P450s) contain numerous channels thought to be populated by their substrates, products, solvents, and gases, yet the principles underlying channel preference are unknown. We identified multiple putative ligand migration channels of two bacterial P450s, CYP102A1 (BM3) from Bacillus megaterium and CYP102A5 from Bacillus cereus, using implicit ligand sampling and free molecular dynamics simulations and furthermore characterized the energy of gas migration through each. We observed strong discrimination between preferred gas migration channels, previously identified substrate/product migration channels and water channels, and mapped putative O 2 reservoirs in the enzyme core. The protein backbone dynamics (S 2 order parameter) unexpectedly revealed that some channels are transient in nature, with subchannels forming and merging and O 2 molecules hopping between subchannels. Finally, we present evidence of the evolution toward O 2 binding in conjunction with protection against inhibitory CO and exclusion of N 2 . Our results significantly enhance our understanding of gas migration in proteins and provide insights into the evolution of gas-utilizing enzymes.
The complexes [(η5-C5R5)Co( i Pr2Im)(η2-C2H4)] (R = H 1; Me 2) were synthesized in good yields via reaction of one equivalent of the N-heterocyclic carbene i Pr2Im (R2Im = 1,3-dialkylimidazolin-2-ylidene) and the bis(ethylene) complexes [(η5-C5R5)Co(η2-C2H4)2]. These complexes serve as convenient starting materials for chemistry using the [(η5-C5R5)Co( i Pr2Im)] complex fragment. The reaction with carbon monoxide leads to the carbonyl complexes [(η5-C5R5)Co( i Pr2Im)(CO)] (R = H 3; Me 4) in good to excellent yields. The carbonyl complexes 3 and 4 are very air sensitive and react readily with oxygen in the solid state and in solution. Whereas the cyclopentadienyl-substituted complex [(η5-C5H5)Co( i Pr2Im)(CO)] (3) decomposes upon reaction with O2 to intractable products, [(η5-C5Me5)Co( i Pr2Im)(CO)] (4) yields the structurally characterized cobalt(III) carbonato complex [(η5-C5Me5)Co( i Pr2Im)(κ2-CO3)] (5). This reaction represents the first example of O2 oxidation of a metal-bound carbonyl for a 3d transition metal complex. The oxidation is too fast to be monitored by NMR spectroscopy, and application of low-temperature time-resolved UV/vis spectroscopy combined with stopped-flow techniques led to the detection of a possible intermediate. On the basis of these experiments and computational investigations using density functional theory (DFT) the peroxo acyl complex [(η5-C5Me5)Co( i Pr2Im)(κ2-C,O-C{O}OO)] (B) is assumed to be the key intermediate detected. The DFT calculations further reveal that this reaction is strongly exothermic with two kinetic barriers, one for the exothermic addition of O2 to the carbonyl complexes 4 to give the peroxo acyl complex [(η5-C5Me5)Co( i Pr2Im)(κ2-C,O-C{O}OO)] (B), the other for the rearrangement of B to give the carbonato complex [(η5-C5Me5)Co( i Pr2Im)(η2-CO3)] (5). The key step for the rearrangement is the formation of CO2 in the coordination sphere of cobalt and the attack of metal-bound oxygen at the carbon atom of CO2.
RNase H is a prototypical example for two metal ion catalysis in enzymes. An RNase H activity cleaving the ribonucleic acid (RNA) backbone of a DNA/RNA hybrid is present in important drug targets, such as the HIV-1 reverse transcriptase but also in many other nucleases such as Homo sapiens (Hs), Escherichia coli (Ec) RNases H or, notably, in enzymes that are part of the CRISPR gene editing molecular machinery. Despite its importance, the reaction mechanism uncovering the proton transfer events are not yet understood. In particular, it is not known, which group is the proton donor for the leaving group. Moreover, several different proton acceptors were proposed, and the exact identity of the proton acceptor is also elusive. Here, we revisit the mechanism for RNAse H, whereby we find that the highly conserved Glu residue of the DDE motif acts as a proton donor via a mechanism further stabilized by the 2'O atom of the sugar. Additionally, we also describe an alternative proton transfer mechanism via a conserved catalytic His residue to deprotonate the attacking water molecule. Furthermore, our quantum mechanics/molecular mechanics (QM/MM) calculations combining Hamiltonian replica exchange with a finite-temperature string method provide an accurate free energy profile for the reaction catalyzed by the HIV-1 RNase H. Our reported pathway is consistent with kinetic data obtained for mutant HIV-1, Hs and Ec RNase H, with the calculated pKa values of the DEDD residues and with crystallographic studies. The overall reaction barrier of ∼19 kcal mol -1 , encountered in the phosphate cleavage step, matches the slow experimental rate of ∼1-100 min -1 . Additionally, using molecular dynamics (MD) calculations, we sample the recently identified binding site for a third transient divalent metal ion in the vicinity of the scissile phosphate in the product complex. Our results account for the experimental observation of a third metal ion facilitating product release in an Aquifex aeolicus RNase III crystal structure and the Bh RNase H in crystallo reaction. Taken together, we provide a molecular mechanism of the nuclease catalytic reaction that is likely common for the broad family of two-metal ion catalytic phosphate cleaving enzymes with a DDE motif.
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