Metal centers in the anaerobic microbial metabolism of CO and CO2

Carbon dioxide and carbon monoxide are important components of the carbon cycle. Major research efforts are underway to develop better technologies to utilize the abundant greenhouse gas, CO(2), for harnessing 'green' energy and producing biofuels. One strategy is to convert CO(2) into CO, which has been valued for many years as a synthetic feedstock for major industrial processes. Living organisms are masters of CO(2) and CO chemistry and, here, we review the elegant ways that metalloenzymes catalyze reactions involving these simple compounds. After describing the chemical and physical properties of CO and CO(2), we shift focus to the enzymes and the metal clusters in their active sites that catalyze transformations of these two molecules. We cover how the metal centers on CO dehydrogenase catalyze the interconversion of CO and CO(2) and how pyruvate oxidoreductase, which contains thiamin pyrophosphate and multiple Fe(4)S(4) clusters, catalyzes the addition and elimination of CO(2) during intermediary metabolism. We also describe how the nickel center at the active site of acetyl-CoA synthase utilizes CO to generate the central metabolite, acetyl-CoA, as part of the Wood-Ljungdahl pathway, and how CO is channelled from the CO dehydrogenase to the acetyl-CoA synthase active site. We cover how the corrinoid iron-sulfur protein interacts with acetyl-CoA synthase. This protein uses vitamin B(12) and a Fe(4)S(4) cluster to catalyze a key methyltransferase reaction involving an organometallic methyl-Co(3+) intermediate. Studies of CO and CO(2) enzymology are of practical significance, and offer fundamental insights into important biochemical reactions involving metallocenters that act as nucleophiles to form organometallic intermediates and catalyze C-C and C-S bond formations.     

Performance and prospects of Rag genes for management of soybean aphid

The soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is an invasive insect pest of soybean [Glycine max (L.) Merr. (Fabaceae)] in North America, and it has led to extensive insecticide use in northern soybean‐growing regions there. Host plant resistance is one potential alternative strategy for managing soybean aphid. Several Rag genes that show antibiosis and antixenosis to soybean aphid have been recently identified in soybean, and field‐testing and commercial release of resistant soybean lines have followed. In this article, we review results of field tests with soybean lines containing Rag genes in North America, then present results from a coordinated regional test across several field sites in the north‐central USA, and finally discuss prospects for use of Rag genes to manage soybean aphids. Field tests conducted independently at multiple sites showed that soybean aphid populations peaked in late summer on lines with Rag1 or Rag2 and reached economically injurious levels on susceptible lines, whereas lines with a pyramid of Rag1 + Rag2 held soybean aphid populations below economic levels. In the regional test, aphid populations were generally suppressed by lines containing one of the Rag genes. Aphids reached putative economic levels on Rag1 lines for some site years, but yield loss was moderated, indicating that Rag1 may confer tolerance to soybean aphid in addition to antibiosis and antixenosis. Moreover, no yield penalty has been found for lines with Rag1, Rag2, or pyramids. Results suggest that use of aphid‐resistant soybean lines with Rag genes may be viable for managing soybean aphids. However, virulent biotypes of soybean aphid were identified before release of aphid‐resistant soybean, and thus a strategy for optimal deployment of aphid‐resistant soybean is needed to ensure sustainability of this technology.     

Evidence that ferredoxin interfaces with an internal redox shuttle in Acetyl-CoA synthase during reductive activation and catalysis

Acetyl-CoA synthase (ACS), a subunit of the bifunctional CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) complex of Moorella thermoacetica requires reductive activation in order to catalyze acetyl-CoA synthesis and related partial reactions, including the CO/[1-(14)C]-acetyl-CoA exchange reaction. We show that the M. thermoacetica ferredoxin(II) (Fd-II), which harbors two [4Fe-4S] clusters and is an electron acceptor for CODH, serves as a redox activator of ACS. The level of activation depends on the oxidation states of both ACS and Fd-II, which strongly suggests that Fd-II acts as a reducing agent. By the use of controlled potential enzymology, the midpoint reduction potential for the catalytic one-electron redox-active species in the CO/acetyl-CoA exchange reaction is -511 mV, which is similar to the midpoint reduction potential that was earlier measured for other reactions involving ACS. Incubation of ACS with Fd-II and CO leads to the formation of the NiFeC species, which also supports the role of Fd-II as a reductant for ACS. In addition to being a reductant, Fd-II can accept electrons from acetylated ACS, as observed by the increased intensity of the EPR spectrum of reduced Fd-II, indicating that there is a stored electron within an "electron shuttle" in the acetyl-Ni(II) form of ACS. This "shuttle" is proposed to serve as a redox mediator during activation and at different steps of the ACS catalytic cycle.     

Economic threshold for soybean aphid (Hemiptera: Aphididae)

Soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), reached damaging levels in 2003 and 2005 in soybean, Glycine max (L.) Merrill, in most northern U.S. states and Canadian provinces, and it has become one of the most important pests of soybean throughout the North Central region. A common experimental protocol was adopted by participants in six states who provided data from 19 yield-loss experiments conducted over a 3-yr period. Population doubling times for field populations of soybean aphid averaged 6.8 d +/- 0.8 d (mean +/- SEM). The average economic threshold (ET) over all control costs, market values, and yield was 273 +/- 38 (mean +/- 95% confidence interval [CI], range 111-567) aphids per plant. This ET provides a 7-d lead time before aphid populations are expected to exceed the economic injury level (EIL) of 674 +/- 95 (mean +/- 95% CI, range 275-1,399) aphids per plant. Peak aphid density in 18 of the 19 location-years occurred during soybean growth stages R3 (beginning pod formation) to R5 (full size pod) with a single data set having aphid populations peaking at R6 (full size green seed). The ET developed here is strongly supported through soybean growth stage R5. Setting an ET at lower aphid densities increases the risk to producers by treating an aphid population that is growing too slowly to exceed the EIL in 7 d, eliminates generalist predators, and exposes a larger portion of the soybean aphid population to selection by insecticides, which could lead to development of insecticide resistance.     

Acute appendicitis in a chimpanzee (Pan troglodytes)

BACKGROUND: A 7-year old, female chimpanzee (Pan troglodytes) developed acute abdominal pain and anorexia. An irregular, mineral opacity was identified in the caudal right quadrant of the abdomen on radiographs and computed tomography scan, which appeared to be in the region of the cecal appendage. RESULTS AND CONCLUSIONS: A diagnosis of acute appendicitis was made based on clinical signs, abnormal haematology findings, and consultation with a human radiologist. Exploratory laparotomy was performed and the cecal appendage was removed. On histologic examination, the mucosal epithelium contained eosinophilic and neutrophilic inflammation. The inflammation extended through the tunica muscularis to the serosal surface and adjacent mesentery. The histologic findings were consistent with acute appendicitis in humans. The chimpanzee recovered well from surgery with immediate improvement in clinical signs and no post-operative complications.     

Protein/Protein Interactions in the Mammalian Heme Degradation Pathway: HEME OXYGENASE-2, CYTOCHROME P450 REDUCTASE,AND BILIVERDIN REDUCTASE*

Heme oxygenase (HO) catalyzes the rate-limiting step in the O₂-dependent degradation of heme to biliverdin, CO, and iron with electrons delivered from NADPH via cytochrome P450 reductase (CPR). Biliverdin reductase (BVR) then catalyzes conversion of biliverdin to bilirubin. We describe mutagenesis combined with kinetic, spectroscopic (fluorescence and NMR), surface plasmon resonance, cross-linking, gel filtration, and analytical ultracentrifugation studies aimed at evaluating interactions of HO-2 with CPR and BVR. Based on these results, we propose a model in which HO-2 and CPR form a dynamic ensemble of complex(es) that precede formation of the productive electron transfer complex. The ¹H-¹⁵N TROSY NMR spectrum of HO-2 reveals specific residues, including Leu-201, near the heme face of HO-2 that are affected by the addition of CPR, implicating these residues at the HO/CPR interface. Alanine substitutions at HO-2 residues Leu-201 and Lys-169 cause a respective 3- and 22-fold increase in K_m values for CPR, consistent with a role for these residues in CPR binding. Sedimentation velocity experiments confirm the transient nature of the HO-2·CPR complex (K_d = 15.1 μm). Our results also indicate that HO-2 and BVR form a very weak complex that is only captured by cross-linking. For example, under conditions where CPR affects the ¹H-¹⁵N TROSY NMR spectrum of HO-2, BVR has no effect. Fluorescence quenching experiments also suggest that BVR binds HO-2 weakly, if at all, and that the previously reported high affinity of BVR for HO is artifactual, resulting from the effects of free heme (dissociated from HO) on BVR fluorescence.     

Radical reactions of thiamin pyrophosphate in 2-oxoacid oxidoreductases

Thiamin pyrophosphate (TPP) is essential in carbohydrate metabolism in all forms of life. TPP-dependent decarboxylation reactions of 2-oxo-acid substrates result in enamine adducts between the thiazolium moiety of the coenzyme and decarboxylated substrate. These central enamine intermediates experience different fates from protonation in pyruvate decarboxylase to oxidation by the 2-oxoacid dehydrogenase complexes, the pyruvate oxidases, and 2-oxoacid oxidoreductases. Virtually all of the TPP-dependent enzymes, including pyruvate decarboxylase, can be assayed by 1-electron redox reactions linked to ferricyanide. Oxidation of the enamines is thought to occur via a 2-electron process in the 2-oxoacid dehydrogenase complexes, wherein acyl group transfer is associated with reduction of the disulfide of the lipoamide moiety. However, discrete 1-electron steps occur in the oxidoreductases, where one or more [4Fe-4S] clusters mediate the electron transfer reactions to external electron acceptors. These radical intermediates can be detected in the absence of the acyl-group acceptor, coenzyme A (CoASH). The π-electron system of the thiazolium ring stabilizes the radical. The extensively delocalized character of the radical is evidenced by quantitative analysis of nuclear hyperfine splitting tensors as detected by electron paramagnetic resonance (EPR) spectroscopy and by electronic structure calculations. The second electron transfer step is markedly accelerated by the presence of CoASH. While details of the second electron transfer step and its facilitation by CoASH remain elusive, expected redox properties of potential intermediates limit possible scenarios. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.