A crystal structure of the anaerobic Ni-Fe-S carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum has been determined to 2.8-Å resolution. The CODH family, for which the R. rubrum enzyme is the prototype, catalyzes the biological oxidation of CO at an unusual Ni-Fe-S cluster called the C-cluster. The Ni-Fe-S C-cluster contains a mononuclear site and a four-metal cubane. Surprisingly, anomalous dispersion data suggest that the mononuclear site contains Fe and not Ni, and the four-metal cubane has the form [NiFe3S4] and not [Fe4S4]. The mononuclear site and the four-metal cluster are bridged by means of Cys 531 and one of the sulfides of the cube. CODH is organized as a dimer with a previously unidentified [Fe4S4] cluster bridging the two subunits. Each monomer is comprised of three domains: a helical domain at the N terminus, an ␣͞␤ (Rossmann-like) domain in the middle, and an ␣͞␤ (Rossmann-like) domain at the C terminus. The helical domain contributes ligands to the bridging [Fe4S4] cluster and another [Fe4S4] cluster, the B-cluster, which is involved in electron transfer. The two Rossmann domains contribute ligands to the active site C-cluster. This x-ray structure provides insight into the mechanism of biological CO oxidation and has broader significance for the roles of Ni and Fe in biological systems. P hototrophic anaerobes such as Rhodospirillum rubrum have the ability to use CO as a sole carbon and energy source (1). This ability derives from the oxidation of CO to CO 2 catalyzed by carbon monoxide dehydrogenases (CODHs). Some CODH enzymes also participate in the synthesis or degradation of acetyl-CoA and are referred to as CODH͞acetyl CoA synthases (CODH͞ACS). The dual role of these CODH͞ACSs is found in CO 2 fixation to acetyl-CoA by acetogens via the well studied Wood͞Ljungdahl pathway (reviewed in ref.2). Methanogens also use CODH͞ACSs in the conversion of acetyl-CoA to methane. As a consequence of these activities, CODHs are essential for the proper regulation of environmental carbon monoxide. Each year, up to 10 8 tons of CO are oxidized to CO 2 by aerobic and anaerobic bacteria containing CODH enzymes (3). Prehistoric environments rich in CO and CO 2 and deficient in oxygen (4) prompt speculation that early life forms relied on CO͞CO 2 as prime sources of both carbon and energy. In an effort to understand the detailed biochemical workings of early and current life on CO, we present the three-dimensional structure of an anaerobic Ni-Fe-S CODH enzyme.CODHs catalyze the oxidation of carbon monoxide in the two-electron process.The mechanism of CO oxidation is thought to involve binding and deprotonation of an H 2 O molecule to form hydroxide at a unique Ni-Fe-S center called the C-cluster (5). CO is believed to bind to a site on the C-cluster adjacent to the hydroxide. Thus, a metal-bound hydroxide may attack the CO carbon. Then the resulting metal-COOH intermediate is deprotonated and CO 2 is lost to yield a two-electron-reduced C-cluster (reviewed in ref.2). In R. rubrum, electrons are pas...

Rho GTPases regulate multiple cellular processes including actin cytoskeletal rearrangements, transcriptional regulation, and oxidant production. The studies described herein demonstrate that small molecule redox agents, in addition to protein regulatory factors, can regulate the activity of redox-active Rho GTPases. A novel (GXXXXGK(S/T)C) motif, conserved in a number of Rho GTPases, appears critical for redox-mediated guanine nucleotide dissociation in vitro. A detailed molecular mechanism for redox regulation of GXXXXGK(S/ T)C motif-containing Rho GTPases is proposed.Rho GTPases comprise a large branch of the Ras superfamily of small guanine nucleotide-binding proteins and include the well characterized family members Rac1, RhoA, and Cdc42 (1). They are involved in regulation of a plethora of cellular processes, including cell morphology, movement, and proliferation (2).The guanine nucleotide bound state of most Rho GTPase family members is regulated by three distinct types of protein modulatory agents, which alter GTPase activity by regulating cycling of the GTPase between inactive GDP and active GTPbound forms (2-4). In particular, guanine nucleotide exchange factors (GEFs) 1 facilitate exchange of GDP with GTP to promote GTPase activation, whereas GTPase-activating proteins deactivate the GTPase protein by stimulating hydrolysis of bound GTP to GDP. Deactivation can also be achieved by association with guanine nucleotide dissociation inhibitors, which prevent membrane association and GDP dissociation. Similar to Ras GTPases, exchange of GDP for GTP leads to a conformational change in the GTPase that greatly enhances affinity to downstream effectors. The interaction between the GTPase and effector leads to activation of GTPase effectormediated signal transduction pathways.A fourth distinct type of modulatory agent has been shown to regulate Ras GTPase activity (5-9). Similar to the action of GEFs, various redox agents, including both reactive oxygen species (ROS) and reactive nitrogen species (RNS), have been shown to stimulate Ras guanine nucleotide dissociation in vitro and up-regulate Ras function in vivo. We have recently elucidated the molecular mechanism by which certain redox agents modulate Ras activity (10 -12). Intriguingly, in addition to Ras, Rho GTPase signaling is sensitive to the presence of ROS and RNS and to the redox state of the cell (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23) EXPERIMENTAL PROCEDURESPreparation of Chemicals and Proteins-The chemicals used for all experiments were of the highest grade unless otherwise noted. Radiolabeled guanine nucleotides ([ 3 H]GDP and [ 3 H]GTP) were diluted with unlabeled guanine nucleotides prior to use, giving ϳ1000 dpm/ M nucleotide. Rac1-(1-177), Rac1 F28L, Rac1 C18S, RhoA-(1-181), and Cdc42-(1-188) proteins were expressed and purified as described previously (34). The final proteins were Ͼ95% pure as determined by SDS-PAGE. The protein concentration was determined by the Bradford method (35). XO was purchased from Sigma.Experimental Conditions ...

Nitric oxide (NO), a highly reactive redox molecule, can react with protein thiols and protein metal centers to regulate a multitude of physiological processes. NO has been shown to promote guanine nucleotide exchange on the critical cellular signaling protein p21Ras (Ras) by S-nitrosylation of a redox-active thiol group (Cys(118)). This increases cellular Ras-GTP levels in vivo, leading to activation of downstream signaling pathways. Yet the process by which this occurs is not clear. Although several feasible mechanisms for protein S-nitrosylation with NO and NO donating have been proposed, results obtained from our studies suggest that Ras can be S-nitrosylated by direct reaction of Cys(118) with nitrogen dioxide (*NO(2)), a reaction product of NO with O(2), via a Ras thiyl-radical intermediate (Ras-S*). Results from our studies also indicate that Ras Cys(118) can be S-nitrosylated by direct reaction of Cys(118) with a glutathionyl radical (GS*), a reaction product derived from homolytic cleavage of S-nitrosoglutathione (GSNO). Moreover, we present evidence that reaction of GS* with Ras generates a Ras-S* intermediate during GSNO-mediated Ras S-nitrosylation. The Ras-S(*) radical intermediate formed from reaction of the Ras thiol with either *NO(2) or GS*, in turn, reacts with NO to complete Ras S-nitrosylation. NO and GSNO modulate Ras activity by promoting guanine nucleotide dissociation from Ras. Our results suggest that formation of the Ras radical intermediate, Ras-S*, may perturb interactions between Ras and its guanine nucleotide substrate, resulting in enhancement of guanine nucleotide dissociation from Ras.

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