Life on carbon monoxide: X-ray structure of Rhodospirillum rubrum Ni-Fe-S carbon monoxide dehydrogenase

Abstract

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...

Structure of the α 2 ε 2 Ni-dependent CO dehydrogenase component of the Methanosarcina barkeri acetyl-CoA decarbonylase/synthase complex

“…Although the asymmetric unit contains only one ␣ heterodimer, a crystallographic twofold axis lies between adjacent ␣-subunits generating the observed ␣ 2 2 oligomer. This arrangement is distinct from that predicted for the 26-Å EM structure of the ␣ 2 2 complex from M. thermophila (14) but is similar to that of related crystal structures of Ni-CODHs from nonmethanogens (8)(9)(10)(11).…”

“…CO dehydrogenase (CODH) activity is associated with the ␣-and -subunits that can be isolated as a 220-kDa ␣ 2 2 component (7). The ␣-subunit exhibits partial sequence homology to the Ni-CODH enzymes from CO-using bacteria and the Ni-CODH domain of acetogenic CODH/ACS enzymes, all of which use a catalytic NiFe 4 S 4 -containing C-cluster to mediate the reversible CODH chemistry (8)(9)(10)(11)(12)(13). The role of the -subunit, however, has been unknown, because it does not evince homology with acetogenic CODH/ACS.…”

“…Toward elucidating their features and unique properties, we have undertaken the structural determination of each of the functional components of the ACDS complex isolated from a methanogen grown on acetate as sole source of methane and reducing power, with the goal of constructing a model for the entire protein. Herein, we describe the structure of the first of these targets, the ␣ 2 2 CO dehydrogenase component [class I CODH/ACDS (19)] from M. barkeri and compare it with the determined structures, namely the Nidependent CODH/ACS (class III) from the acetogen M. thermoacetica (10,11) and the Ni-CODH (class IV) from the phototrophic bacteria C. hydrogenoformans (8) and Rhodospirillum rubrum (9). These related Ni-CODHs have been shown to share a similar active site but exhibit significant differences in their global sequence and overall oligomerization.…”

Evidence for a Proton Transfer Network and a Required Persulfide-Bond-Forming Cysteine Residue in Ni-Containing Carbon Monoxide Dehydrogenases

“…However, the partially required nature of this residue suggests that another group might serve the same role. Lys587 is located near to the [Ni Fe] subsite of the C-cluster, and has been proposed to stabilize the Ni-bound carboxylate intermediate (4). The partial loss of activity in the mutant with this residue replaced with Ala is consistent with this proposal if the caveat that another group exists which plays a redundant role is included.…”

The rocky roots of the acetyl-CoA pathway

“…The significance of metal sulphides in the hydrothermal reactor model is their catalytic activity in CO 2 -and COreducing reactions, both as metal sulphur clusters in proteins [16,28,29] (Figure 2) and as the free minerals themselves [24,25]. The significance of the 3D compartments comprising the reactor (Figures 1 and 3), which form a barrier to diffusion into the ocean, is their retention of just-synthesized organic molecules.…”