The Nickel Enzymes

Urease is famous in enzymology for being the first enzyme to be purified and crystallized (1926). At the time enzymes were widely viewed as being too ill-defined for detailed chemical study. Sumner argued that its crystalline character meant that urease was a single defined substance and the fact that he could not find any cofactors led to the conclusion that polypeptides could have catalytic activity on their own. The existence of two essential Ni + ions 

The archaebacteria are very rich in nickel-containing enzymes, and coenzymes, and Nature has clearly chosen this element to bring about the initial steps in the biochemical utilization of H2, CO, CH4, and other C, compounds, at least in an anaerobic environment. These steps almost certainly involve organonickel chemistry, although how this happens in detail is only just beginning to be understood. 

An important detail emphasizes the difference in the behavior of enzymes and of simple compounds although the NiFeC site always contains a full mole of nickel, the epr integration suggested only 0.1-0.3S spins were present per cluster. The addition of phenanthrene (phen) removes 0.1-0.35 Ni per enzyme and abolishes 100% of both the epr signal and the acetyl CoA synthase activity. Although the protein is pure by all the usual criteria, some of the enzyme molecules appear to contain catalytically- and epr-inactive NiFeC clusters in which the Ni cannot be removed by phen. Biological systems are not always so well defined as chemical compounds. 

A fully functional model for the second Ni in CODH has been found 16.17. This complex has the appropriate metal, NF+, as well as an N-, 0-, S-ligand environment and catalyzes the reaction shown in Eq. 16.37. The CO2 is detected by precipitation with Ca(OH)2, the H production with a pH meter, and the electrons formed are transferred to the electron acceptor (16.18) and gives the dark blue radical anion, MV . The reaction probably 

A stoichiometric model system by Holm for the acetylCoA synthase activity of CODH is shown in Eq. 16.41. This reaction is a property of the NiFeC cluster, of unknown structure, present in CODH. The enzyme brings about exchange between CO and Me COCoA, which implies that formation of the C—S and Me—CO bonds is reversible. This is consistent with CO insertion into a Ni—Me bond, and nucleophilic attack on the resulting Ni(COMe), both of which can be reversible. 

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Superoxide dismutases may contain a range of metals Mn, Fe, or both Cu and Zn, and representatives of all these are found in prokaryotes. The nickel enzyme is noted later. 

If E. coli is grown in a cadmium-containing, zinc-deficient medium, the enzyme is found to be active, but to contain six Cd2+ per molecule. The presence of cysteinyl ligands is confirmed by the observation of the characteristic charge-transfer bands. The binding of substrate perturbs the absorption and CD spectra of the zinc and cadmium enzymes, and the d-d spectrum of the nickel enzyme, showing that "the conformation of the R subunit is affected by the binding of substrate to the C subunit.530,531... 

The nickel enzymes covered in this article can be divided into two groups redox enzymes and hydrolases. The five Ni redox enzymes are hydrogenase, CO dehydrogenase (CODH), acetyl-CoA synthase (ACS), methyl-Coenzyme M reductase (MCR), and superoxide dismutase (SOD). Glyoxalase-I and urease are Ni hydrolases. Ni proteins that are not enzymes are not covered, because they have been recently reviewed. These include regulatory proteins (NikR) and chaperonins and metal uptake proteins (CooJ, CooE, UreE, and ABC transporters). A recent crystal structure of NikR, shown in Figure l(i), is a notable recent achievement in this area. ... 

Chemistry relating to the nickel enzymes CODH and ACS 05CCR(249)1582. 

All the systems described in this chapter are organometallic in character. Coenzyme has several forms with M—C or M—H bonds. In nitrogen fixation, CO binds competitively at the active site. The nickel enzymes are believed to operate via intermediates with M—H (H2ase) or M—C bonds (CODH and MeCoM reductase). 

Methanogene enzymes reduce CO2 to CH4 and recover the resulting energy. In the last step, methyl-coenzyme M is hydrogenated to methane by a thiol cofactor HS-HTP, the reaction being catalyzed by the nickel enzyme. 

A coenzyme containing a Ni-methanocorphin complex might be bound to the nickel enzyme and would contain the active site allowing the transformation of the methyl group. 

The topics covered here have an organometallic connection. Coenzyme Bi2 has M-C or M-H bonds, and the active site cluster in nitrogen flxation has a carbon atom at its heart. The nickel enzymes go... 

Until the discovery in 1975 of nickel in jack bean urease (which, 50 years previously, had been the first enzyme to be isolated in crystalline form and was thought to be metal-free) no biological role for nickel was known. Ureases occur in a wide variety of bacteria and plants, catalyzing the hydrolysis of urea,... 

CODH/ACS is an extremely oxygen-sensitive protein that has been found in anaerobic microbes. It also is one of the three known nickel iron-sulfur proteins. Some authors would consider that there are only two, since the CODH and ACS activities are tightly linked in many organisms. However, there is strong evidence that the ACS and CODH activities are associated with different protein subunits and the reactions that the two enzymes catalyze are quite different. CODH catalyzes a redox reaction and ACS catalyzes the nonredox condensation of a methyl group, a carbonyl group, and an organic thiol (coenzyme A). 

Fe Q-band ENDOR study of the isotopically enriched Ni-C state of D. gigas and D. desulfuricans hydrogenases and Ni-B state of D. desulfuricans revealed a weak coupling between the Fe and the nickel atoms when the enzyme was in the Ni-A forms while no coupling was observed for the Ni-B form (186). A careful analysis of linewidth of Ni-A and Ni-B EPR signals detected in Fe enriched and nonenriched hydrogenase samples indicated that hyperfine interactions are lost in the spectral linewidth and, hence, nonde-tectable. 

The discovery of a new heterodinuclear active site in [NiFe] hydro-genases opens the way for the proposal of catalytic cycles based on the available spectroscopic data on the different active site redox states, namely EXAFS studies that reveal that the Ni-edge energy upon reduction of the enzyme supports an increase in the charge density of the nickel (191). 

Nickel exists in the tunicate Trididemnum solidum as the nickel complex of a modified chlorin (Bible et al. 1988) and is a component of a number of enzymes. Urease is the classic example of a nickel-containing enzyme, and several enzymes contain both nickel and iron. Details of enzymes that contain nickel have been provided in a review (Mulrooney and Hausinger 2003), and only brief summaries are provided ... 

Methyl coenzyme M reductase plays a key role in the production of methane in archaea. It catalyzes the reduction of methyl-coenzyme M with coenzyme B to produce methane and the heterodisulfide (Figure 3.35). The enzyme is an a2P2Y2 hexamer, embedded between two molecules of the nickel-porphinoid F jg and the reaction sequence has been delineated (Ermler et al. 1997). The heterodisulfide is reduced to the sulfides HS-CoB and HS-CoM by a reductase that has been characterized in Methanosarcina thermoph-ila, and involves low-potential hemes, [Fe4S4] clusters, and a membrane-bound metha-nophenazine that contains an isoprenoid chain linked by an ether bond to phenazine (Murakami et al. 2001). 

A handbook on inorganic and coordination chemistry of porphyrins has been published.1765 Factor F430 is the nickel-hydrocorphinoid group of the enzyme methyl coenzyme M reductase.47,48 The mystery of this particular metalloprotein is one of the major reasons for the development of Ni11-porhyrin coordination chemistry, although not the only one. 

One limitation to this method should be noted. If the antibody-enzyme conjugate is prepared using antibody fragments such as Fab or F(ab )2, then nickel-chelate affinity chromatography will not work, since the requisite Fc portion of the antibody necessary for complexing with the metal is not present. 

Figure 20.15 An affinity chromatography support containing iminodiacetic acid groups chelated with nickel may be used to remove excess enzyme after reactions to produce antibody-enzyme conjugates. The nickel chelate binds to the antibody in the Fc region, retaining the conjugate while allowing free enzyme to pass through the gel unretarded.

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