Protein dynamics in the reductive activation of a B12-containing enzyme

B12-dependent proteins are involved in methyl transfer reactions ranging from the biosynthesis of methionine in humans to the formation of acetyl-CoA in anaerobic bacteria. During their catalytic cycle, they undergo large conformational changes to interact with various proteins. Recently, the crystal structure of the B12-containing corrinoid iron–sulfur protein (CoFeSP) in complex with its reductive activator (RACo) was determined, providing a first glimpse of how energy is transduced in the ATP-dependent reductive activation of corrinoid-containing methyltransferases. The thermodynamically uphill electron transfer from RACo to CoFeSP is accompanied by large movements of the cofactor-binding domains of CoFeSP. To refine the structure-based mechanism, we analyzed the conformational change of the B12-binding domain of CoFeSP by pulsed electron–electron double resonance and Förster resonance energy transfer spectroscopy. We show that the site-specific labels on the flexible B12-binding domain and the small subunit of CoFeSP move within 11 Å in the RACo:CoFeSP complex, consistent with the recent crystal structures. By analyzing the transient kinetics of formation and dissociation of the RACo:CoFeSP complex, we determined values of 0.75 μM–1 s–1 and 0.33 s–1 for rate constants kon and koff, respectively. Our results indicate that the large movement observed in crystals also occurs in solution and that neither the formation of the protein encounter complex nor the large movement of the B12-binding domain is rate-limiting for the ATP-dependent reductive activation of CoFeSP by RACo

GAIT ANALYSIS OF THE NORMAL AND ACL DEFICIENT PATIENTS AFTER LIGAMENT RECONSTRUCTION SURGERY

Anterior cruciate ligament (ACL) injury of the knee is common in sports. A serious ACL injury leads to ligament reconstruction surgery. In order to evaluate result of surgery or optimize the rehabilitation process, a knee condition must be objectively identified. The purpose of this study is, therefore, to numerically indicate and classify knee condition of patients via the chaos analysis. Lyapunov exponents (LyEs) were used for the comparison of the normal and the patients

A Morphing [4Fe-3S-nO]-Cluster within a Carbon Monoxide Dehydrogenase Scaffold

Ni,Fe-containing carbon monoxide dehydrogenases (CODHs) catalyze the reversible reduction of CO2 to CO. Several anaerobic microorganisms encode multiple CODHs in their genome, of which some, despite being annotated as CODHs, lack a cysteine of the canonical binding motif for the active site Ni,Fe-cluster. Here, we report on the structure and reactivity of such a deviant enzyme, termed CooS-VCh. Its structure reveals the typical CODH scaffold, but contains an iron-sulfur-oxo hybrid-cluster. Although closely related to true CODHs, CooS-VCh catalyzes neither CO oxidation, nor CO2 reduction. The active site of CooS-VCh undergoes a redox-dependent restructuring between a reduced [4Fe-3S]-cluster and an oxidized [4Fe-2S-S*-2O-2(H2O)]-cluster. Hydroxylamine, a slow-turnover substrate of CooS-VCh, oxidizes the hybrid-cluster in two structurally distinct steps. Overall, minor changes in CODHs are sufficient to accommodate a Fe/S/O-cluster in place of the Ni,Fe-heterocubane-cluster of CODHs

Complete Genome Sequence of Brucella abortus A13334, a New Strain Isolated from the Fetal Gastric Fluid of Dairy Cattle

Brucella abortus is a major pathogen that infects livestock and humans. A new strain of B. abortus (A13334) was isolated from the fetal gastric fluid of a dairy cow, with the aim of using it to compare genetic properties, analyze virulence factor, and survey the epidemiological relationship to other Brucella species. Here, we report the complete and annotated genome sequence of B. abortus A13334.open2

Complete Genome Sequence of Brucella canis Strain HSK A52141, Isolated from the Blood of an Infected Dog

Brucella canis infection can be clinically inapparent in dogs, and when infection goes unnoticed, there is a chance for dog-to-human transmission. A new strain of B. canis was isolated from the blood of an infected dog in order to analyze the pathogenic mechanism, compare genetic properties, and develop new genetic tools for early diagnosis of canine brucellosis. Herein, we report the complete genome sequence of the strain B. canis HSK A52141. This is the second complete genome sequence and biological annotation available for a member of B. canis.open2

Double-Cubane [8Fe9S] Clusters: A Novel Nitrogenase-Related Cofactor in Biology

Three different types of electron‐transferring metallo‐ATPases are able to couple ATP hydrolysis to the reduction of low‐potential metal sites, thereby energizing an electron. Besides the Fe‐protein known from nitrogenase and homologous enzymes, two other kinds of ATPase with different scaffolds and cofactors are used to achieve a unidirectional, energetic, uphill electron transfer to either reduce inactive Co‐corrinoid‐containing proteins (RACE‐type activators) or a second iron‐sulfur cluster‐containing enzyme of a unique radical enzymes family (archerases). We have found a new cofactor in the latter enzyme family, that is, a double‐cubane cluster with two [4Fe4S] subclusters bridged by a sulfido ligand. An enzyme containing this cofactor catalyzes the ATP‐dependent reduction of small molecules, including acetylene. Thus, enzymes containing the double‐cubane cofactor are analogous in function and share some structural features with nitrogenases.Peer Reviewe

Stepwise O2‐Induced Rearrangement and Disassembly of the [NiFe4(OH)(μ3‐S)4] Active Site Cluster of CO Dehydrogenase

Ni,Fe‐containing carbon monoxide dehydrogenases (CODHs) catalyze the reversible reduction of carbon dioxide to carbon monoxide. CODHs are found in anaerobic microorganisms and can rapidly lose their activity when exposed to air. What causes the loss of activity is unclear. In this study, we analyzed the time‐dependent structural changes induced by the presence of air on the metal centers of CODH‐II. We show that inactivation is a multistep process. In a reversible step, the open coordination site on the Ni ion is blocked by a Ni,Fe‐bridging μ‐sulfido or chlorido ligand. Blocking this open coordination site with a cyanide ligand stabilizes the cluster against O2‐induced decomposition, indicating that O2 attacks at the Ni ion. In the subsequent irreversible phase, nickel is lost, the Fe ions rearrange and the sulfido ligands disappear. Our data are consistent with a reversible reductive reactivation mechanism to protect CODHs from transient over‐oxidation.Peer Reviewe

A Morphing [4Fe-3S-nO]-Cluster within a Carbon Monoxide Dehydrogenase Scaffold

Ni,Fe-containing carbon monoxide dehydrogenases (CODHs) catalyze the reversible reduction of CO2 to CO. Several anaerobic microorganisms encode multiple CODHs in their genome, of which some, despite being annotated as CODHs, lack a cysteine of the canonical binding motif for the active site Ni,Fe-cluster. Here, we report on the structure and reactivity of such a deviant enzyme, termed CooS-VCh. Its structure reveals the typical CODH scaffold, but contains an iron-sulfur-oxo hybrid-cluster. Although closely related to true CODHs, CooS-VCh catalyzes neither CO oxidation, nor CO2 reduction. The active site of CooS-VCh undergoes a redox-dependent restructuring between a reduced [4Fe-3S]-cluster and an oxidized [4Fe-2S-S*-2O-2(H2O)]-cluster. Hydroxylamine, a slow-turnover substrate of CooS-VCh, oxidizes the hybrid-cluster in two structurally distinct steps. Overall, minor changes in CODHs are sufficient to accommodate a Fe/S/O-cluster in place of the Ni,Fe-heterocubane-cluster of CODHs.Peer Reviewe

Substrate Activation at the Ni,Fe Cluster of CO Dehydrogenases: The Influence of the Protein Matrix

Carbon monoxide dehydrogenases catalyze the reversible conversion of CO2 with two electrons to CO and water at a unique Ni- and Fe-containing cluster (cluster C). Structural studies indicate that several highly conserved amino acids in the second coordination sphere of cluster C support the activation of the substrates, CO/CO2 and water, and may be mandatory for catalytic turnover. However, their contribution to substrate activation has been poorly explored. We replaced the three residues with potential direct interaction with the substrates (I567, H93, and K563) and one residue essential for proton/water transfer (H96) and analyzed the associated changes in the structure and reactivity of the enzyme. In addition to the expected exchange of side chains, we observed rearrangements of water molecules as well as the appearance of additional water molecules at the active site. These changes also affect the coordination of cluster C and the hydroxo ligand at Fe, with additional hydroxo/water ligands at Ni. Subsequently, we were able to convert cluster C from a [NiFe4(OH)(μ3-S)4] cluster to a [Fe4(μ3-S)4] cluster by exchanging K563 and a primary coordinating C295. Therefore, the second coordination sphere is important not only for the affinity of the substrates but also for the stability of cluster C. Thus, beyond substrate activation, the residues in the second coordination sphere of cluster C also determine its coordination and stability