An alcove at the acetyl-CoA synthase nickel active site is required for productive substrate CO binding and anaerobic carbon fixation.

One of seven natural CO2 fixation pathways, the anaerobic Wood-Ljungdahl Pathway (WLP) is unique in generating CO as a metabolic intermediate, operating through organometallic intermediates, and in conserving (versus utilizing) net ATP. The key enzyme in the WLP is acetyl-CoA synthase (ACS), which uses an active site [2Ni-4Fe-4S] cluster (A-cluster), a CO tunnel, and an organometallic (Ni-CO, Ni-methyl, and Ni-acetyl) reaction sequence to generate acetyl-CoA. Here we reveal that an alcove, which interfaces the tunnel and the A-cluster, is essential for CO2 fixation and autotrophic growth by the WLP. In vitro spectroscopy, kinetics, binding, and in vivo growth experiments reveal that a Phe229A substitution at one wall of the alcove decreases CO affinity thirty-fold and abolishes autotrophic growth; however, a F229W substitution enhances CO binding 80-fold. Our results indicate the structure of the alcove is exquisitely tuned to concentrate CO near the A-cluster; protect ACS from CO loss during catalysis, provide a haven for inhibitory CO, and stabilize the tetrahedral coordination at the Nip site where CO binds. The directing, concentrating, and protective effects of the alcove explain the inability of F209A to grow autotrophically. The alcove also could help explain current controversies over whether ACS binds CO and methyl through a random or ordered mechanism. Our work redefines what we historically refer to as the metallocenter "active site". The alcove is so crucial for enzymatic function that we propose it is part of the active site. The community should now look for such alcoves in all "gas handling" metalloenzymes.

Rapid-reaction kinetics of the bifurcating NAD+-dependent NADPH:ferredoxin oxidoreductase NfnI from Pyrococcus furiosus.

We have investigated the kinetics of NAD+-dependent NADPH:ferredoxin oxidoreductase (NfnI), a bifurcating transhydrogenase that takes two electron pairs from NADPH to reduce two ferredoxins and one NAD+ through successive bifurcation events. NADPH reduction takes place at the bifurcating FAD of NfnI's large subunit, with high-potential electrons transferred to the [2Fe-2S] cluster and S-FADH of the small subunit, ultimately on to NAD+; low-potential electrons are transferred to two [4Fe-4S] clusters of the large subunit and on to ferredoxin. Reduction of NfnI by NADPH goes to completion only at higher pH, with a limiting kred of 36 ± 1.6 s-1 and apparent KdNADPH of 5 ± 1.2 µM. Reduction of one of the [4Fe-4S] clusters of NfnI occurs within a second, indicating that in the absence of NAD+, the system can bifurcate and generate low-potential electrons without NAD+. When enzyme is reduced by NADPH in the absence of NAD+ but the presence of ferredoxin, up to three equivalents of ferredoxin become reduced, although the reaction is considerably slower than seen during steady-state turnover. Bifurcation appears to be limited by transfer of the first, high-potential electron into the high-potential pathway. Ferredoxin reduction without NAD+ demonstrates that electron bifurcation is an intrinsic property of the bifurcating FAD and is not dependent on the simultaneous presence of NAD+ and ferredoxin. The tight coupling between NAD+ and ferredoxin reduction observed under multiple-turnover conditions is instead simply due to the need to remove reducing equivalents from the high-potential electron pathway under multiple-turnover conditions.

Characterization of Methyl- and Acetyl-Ni Intermediates in Acetyl CoA Synthase Formed during Anaerobic CO2 and CO Fixation.

The Wood-Ljungdahl Pathway is a unique biological mechanism of carbon dioxide and carbon monoxide fixation proposed to operate through nickel-based organometallic intermediates. The most unusual steps in this metabolic cycle involve a complex of two distinct nickel-iron-sulfur proteins: CO dehydrogenase and acetyl-CoA synthase (CODH/ACS). Here, we describe the nickel-methyl and nickel-acetyl intermediates in ACS completing the characterization of all its proposed organometallic intermediates. A single nickel site (Nip) within the A cluster of ACS undergoes major geometric and redox changes as it transits the planar Nip, tetrahedral Nip-CO and planar Nip-Me and Nip-Ac intermediates. We propose that the Nip intermediates equilibrate among different redox states, driven by an electrochemical-chemical (EC) coupling process, and that geometric changes in the A-cluster linked to large protein conformational changes control entry of CO and the methyl group.

13C Electron Nuclear Double Resonance Spectroscopy Shows Acetyl-CoA Synthase Binds Two Substrate CO in Multiple Binding Modes and Reveals the Importance of a CO-Binding "Alcove".

EPR and Electron Nuclear Double Resonance spectroscopies here characterize CO binding to the active-site A cluster of wild-type (WT) Acetyl-CoA Synthase (ACS) and two variants, F229W and F229A. The A-cluster binds CO to a proximal Ni (Nip) that bridges a [4Fe-4S] cluster and a distal Nid. An alcove seen in the ACS crystal structure near the A-cluster, defined by hydrophobic residues including F229, forms a cage surrounding a Xe mimic of CO. Previously, we only knew WT ACS bound a single CO to form the Ared-CO intermediate, containing Nip(I)-CO with CO located on the axis of the dz2 odd-electron orbital (g⊥ > g|| ∼ 2). Here, the two-dimensional field-frequency pattern of 2K-35 GHz 13C-ENDOR spectra collected across the Ared-CO EPR envelope reveals a second CO bound in the dz2 orbital's equatorial plane. This WT A-cluster conformer dominates the nearly conservative F229W variant, but 13C-ENDOR reveals a minority "A" conformation with (g|| > g⊥ ∼ 2) characteristic of a "cloverleaf" (e.g., dx2-y2) odd-electron orbital, with Nip binding two, apparently "in-plane" CO. Disruption of the alcove through introduction of the smaller alanine residue in the F229A variant diminishes conversion to Ni(I) ∼ 10-fold and introduces extensive cluster flexibility. 13C-ENDOR shows the F229A cluster is mostly (60%) in the "A" conformation but with ∼20% each of the WT conformer and an "O" state in which dz2 Nip(I) (g⊥ > g|| ∼ 2) surprisingly lacks CO. This paper thus demonstrates the importance of an intact alcove in forming and stabilizing the Ni(I)-CO intermediate in the Wood-Ljungdahl pathway of anaerobic CO and CO2 fixation.

Time-Resolved Infrared Spectroscopy Reveals the pH-Independence of the First Electron Transfer Step in the [FeFe] Hydrogenase Catalytic Cycle.

[FeFe] hydrogenases are highly active catalysts for hydrogen conversion. Their active site has two components: a [4Fe-4S] electron relay covalently attached to the H2 binding site and a diiron cluster ligated by CO, CN-, and 2-azapropane-1,3-dithiolate (ADT) ligands. Reduction of the [4Fe-4S] site was proposed to be coupled with protonation of one of its cysteine ligands. Here, we used time-resolved infrared (TRIR) spectroscopy on the [FeFe] hydrogenase from Chlamydomonas reinhardtii (CrHydA1) containing a propane-1,3-dithiolate (PDT) ligand instead of the native ADT ligand. The PDT modification does not affect the electron transfer step to [4Fe-4S]H but prevents the enzyme from proceeding further through the catalytic cycle. We show that the rate of the first electron transfer step is independent of the pH, supporting a simple electron transfer rather than a proton-coupled event. These results have important implications for our understanding of the catalytic mechanism of [FeFe] hydrogenases and highlight the utility of TRIR.

Injection Techniques for Common Chronic Pain Conditions of the Foot: A Comprehensive Review.

PURPOSE OF REVIEW: This is a comprehensive literature review of the available evidence and techniques of foot injections for chronic pain conditions. It briefly describes common foot chronic pain syndromes and then reviews available injection techniques for each of these syndromes, weighing the available evidence and comparing the available approaches. RECENT FINDINGS: Foot and ankle pain affects 20% of the population over 50 and significantly impairs mobility and ability to participate in activities of daily living (ADLs), as well as increases fall risk. It is commonly treated with costly surgery, at times with questionable efficacy. Injection therapy is challenging when the etiology is anatomical or compressive. Morton's neuroma is a budging of the interdigital nerve. Steroid, alcohol, and capsaicin injections provide some benefit, but it is short lived. Hyaluronic acid (HA) injection provided long-term relief and could prove to be a viable treatment option. Achilles tendinopathy (AT) is most likely secondary to repeat tendon stress-platelet-rich-plasma (PRP) and prolotherapy have been trialed for this condition, but more evidence is required to show efficacy. Similar injections were trials for plantar fasciitis and achieved only short-term relief; however, some evidence suggests that PRP injections reduce the frequency of required therapy. Tarsal tunnel syndrome, a compressive neuropathy carries a risk of permanent neural injury if left untreated. Injection therapy can provide a bridge to surgery; however, surgical decompression remains the definitive therapy. When the etiology is inflammatory, steroid injection is more likely to provide benefit. This has been shown in several studies for gout, as well as osteoarthritis of the foot and ankle and treatment-refractory rheumatoid arthritis. HA showed similar benefit, possibly due to anti-inflammatory effects. Stem cell injections may provide the additional benefit of structure restoration. Chronic foot pain is common in the general population and has significant associated morbidity and disability. Traditionally treated with surgery, these are costly and only somewhat effective. Injections provide an effective alternative financially and some evidence exists that they are effective in pain alleviation. However, current evidence is limited and the benefit described from injection therapy has been short-lived in most cases. Further studies in larger populations are required to evaluate the long-term effects of these treatments.

Production and properties of enzymes that activate and produce carbon monoxide.

The chapter focuses on the methods involved in producing and characterizing two key nickel-iron-sulfur enzymes in the Wood-Ljungdahl pathway (WLP) of anaerobic conversion of carbon dioxide fixation into acetyl-CoA: carbon monoxide dehydrogenase (CODH) and acetyl-CoA synthase (ACS). The WLP is used for biosynthesis of cell material and energy conservation by anaerobic bacteria and archaea, and it is central to several industrial biotechnology processes aimed at using syngas and waste gases for the production of fuels and chemicals. The pathway can run in reverse to allow organisms, e. g., methanogens and sulfate reducers, to grow on acetate. The CODH and ACS intertwine to form a tenacious CODH/ACS complex that converts CO2, a methyl group, and coenzyme A into acetyl-CoA. CODH also behaves as a modular unit that can function as an independent homodimer. Besides coupling to ACS, CODH can interact with hydrogenases to couple CO oxidation to H2 formation. These enzymes have been purified and characterized from several microbes.