Role Of Metal Ions In Biochemistr

Role Of Metal Ions In Biochemistr A metal is a chemical element that is a good conductor of both electricity and heat and forms cations and ionic bonds with non-metals. In chemistry, ametal (from Greek ÃŽÂ ¼ÃƒÅ½Ã‚ ­Ãƒ Ã¢â‚¬Å¾ÃƒÅ½Ã‚ ±ÃƒÅ½Ã‚ »ÃƒÅ½Ã‚ »ÃƒÅ½Ã‚ ¿ÃƒÅ½Ã‚ ½ mà ©tallon, mine]) is an element, compound, or alloy characterized by high electrical conductivity. In a metal, atoms readily lose electrons to form positive ions (cations). Those ions are surrounded by delocalized electrons, which are responsible for the conductivity. The solid thus produced is held by electrostatic interactions between the ions and the electron cloud, which are called metallic bonds.[2] Metal ions play essential roles in about one third ofenzymes . These ions can modify electron flow I a substrate or enzyme, thus effectively controlling an enzyme-catalyzed reaction. They can serve to bind and orient substrate with respect to functional groups in the active site, and they can provide a site for redox activity if the metal has several valence states. Without the appropriate metal ion, a biochemical reaction catalyzed by a particular metalloenzyme would proceed very slowly, if at all. The enzyme provides an arrangement of sidechain functional groups having an appropriate sized hole with the preferred groups on enzyme side chains needed to bind the required metal ion. The optimal number of such binding groups is chosen for the particular metal ion, together with the appropriate hydrophobic or hydrophilic environment in the binding site. Metal ions may be bound by main-chain amino and carbonyl groups, but specific binding is achieved by the amino acid side chains, particularly the carboxylate groups of aspartic and glutamic acid, and the ring nitrogen atom of histidine. Other side chains that bind metals ions include tryptophan (ring nitrogen), cysteine (thiol), methionine (thioether), serine, threonine, tyrosine (hydroxyl groups), and asparagine and glutamine (carbonyl groups, less often amino group . No set of general rules exists that describes how a given metal ion will behave in an enzyme . Now that many crystal structures of proteins are being studied by X-ray diffraction, information on the binding of metal ions in the active sites of enzymes is available and should provide clues to the mechanism of action of the enzyme.The examples of catechol methyltransferase andmandelate racemase will be discussed later in this article.The work described here includes results fromexaminations of the crystal structures in the CambridgeStructural Database and the Protein Databank . Astudy of binding, however, also involves an analysis ofthe energetic consequences of changing the way thebinding occurs, so that the most stable binding pattern fora given group of ligands can be deduced. We haveapproached this using ab initio molecular orbital and density functional calculations . In this way weobtain both the binding geometry of ligands and theenergetic consequences of changing this binding m ode. Properties of metal ions Metal ions are generally positively charged and act as electrophiles, seeking the possibility of sharing electron pairs with other atoms so that a bond or charge-charge interaction can be formed. They behave rather like hydrogen ions (the poor mans metal). Metal ions, however, often have positive charges greater than one,and have a larger ionic volume so that they can accommodate many ligands around them at the same time. In addition, metal ion concentrations can be high atneutral pH values, while hydrogen ion concentrations are, by the definition of pH, low at these values. Ligands are the atoms or groups of atoms that are bonded to the metal ion, generally in an electrostatic manner. They are usually neutral or negatively charged and they donate electron density to the metal ion. Thecoordination number of a metal ion, that is, the number of ligand atoms bound to it, is viewed in terms of concentric spheres; the inner sphere containing those atoms in contact with the metal ion, the second sphere containing those in contact with the inner sphere ligand atoms. The number of atoms in these spheres will depend on the size of the metal ion and the sizes of the ligand atoms. For example, sodium is smaller than potassium, and sulfur is larger than oxygen. Measurements of metal ion-liganddistances in crystal structures led to the idea of atomic and ionic radii [9-11]; anion radii can also be derived from the minimum anion-anion distances in crystal structures. The radius ratio, a concept introduced by Goldschmidt [11], is the ratio of the radius of the cation to that of the anion and is generally less than 1.0 Tetrahedral structures have a radius ratio between 0.225 and 0.414, while octahedral structures have a ratio between 0.414 and 0.645. For example, the radius of Mg2+ is 0.65 D, while that of O2- is 1.40 D and their radius ratio is 0.464; the packing is octahedral. The charge distribution in the active site of an enzyme is designed to stabilize the transition state of the catalyzed reaction relative to that of the substrate. In enzyme-catalyzed reactions it is essential that the reactants be brought together with the correct spatial orientation, otherwise the chance of the reaction taking place is diminished and the reaction rate will be too low.The electrostatic environment in the active site is a major factor that serves to guide the substrate to the binding site in the correct orientation. Metal ions can assist in this process, often binding groups in a stereochemically rigid manner, thereby helping to control the action of the enzyme. Thus, an enzyme will bind its substrate in such a manner that immobilization and alignment, ready formation of the transition state of the reaction to be catalyzed,and then easy release of the product will result; metal ions often help in accomplishing this process. Each metal ion has its own chemistry. An example of the differing reactivities of metal cations is provided by their ability to bind or lose water molecules. The exchange of coordinated water with bulk solvent by various cations has been categorized into four groups: those for which the exchange rate is greater than 108 per second including alkali and alkaline earth metal ions(except beryllium and magnesium), together with Cr3+,Cu2+, Cd2+, and Hg2+. Intermediate rate constants (from 104 to 108 per second) are found for Mg2+ and some of the divalent first-row transition metal ions. Those with slow rate constants (from 1 to 104 per second) include Be2+ and certain trivalent first-row transition metal ions. The inert group with rates from 10-6 to 10-2 per second containsCr3+, Co3+, Rh3+, Ir3+, and Pt2+. One of the factors involved in rates of exchange is the charge-to-radius Ratio; if this ratio is high the exchange rate is low.An important reaction catalyzed by metal ions inenzymes is the ionization of water to give a hydrated hydrogen ion and a hydroxyl anion. Initial studies of this process will be discussed here as they are relevant to the action of a metal ion in providing a hydroxyl group and a hydrogen ion for use in an enzymatic reaction. Polarizing Potential of Various Ions Atoms or groups of atoms are considered polarizable if, when they are placed in an electric field, a charge separation occurs and a dipole is acquired. This deformability or polarizability is measured by the ratio of the induced dipole to the applied field. Those atoms that hold on less firmly to their electrons are termed more polarizable. It is found that if two ions have the same inert gas structure (potassium and chloride, for example), the negatively charged anion is more polarizable than the positively charged cation, which holds on to its electrons more tightly. The word hard has been introduced to indicate a low polarizability so that the electron cloud is difficult to deform (like a hard sphere). By contrast soft means high polarizability so that the electron cloud is readily deformed . A hard acid or metal cation holds tightly to its electrons and therefore its electron cloud is not readily distorted; its unshared valence electrons are not easily excited. Soft (polarizable) metal cations contain electrons that are not so tightly held and therefore are easily distorted or removed. A hard acid prefers tocombine with a hard base, while a soft acid prefers to bind with a soft base by partially forming covalent bonds .The type of binding is related to the highest occupied molecular orbital (HOMO) of the electron-pair donor (a lewis base, the ligand) and the lowest unoccupied molecular orbital (LUMO) of the electron-pair acceptor (a Lewis acid, the metal ion). If these have similar energies, then electron transfer will give a covalent (soft) interaction, whereas the energy difference is large, electron transfer does not readily take place and the interaction is mainly electrostatic (hard-hard). Hardcations include the alkali and alkaline earth metal ions while soft metal ions include Cu 2+, Hg2 2+, Hg2+, Pd2+. Inbiological systems, hard ligands generally contain oxygen while soft ligands contain sulfur. Hard acids tend to bind hard bases by ionic forces, while soft acids bind soft bases by partially forming covalent bonds. These hard-soft categorizations are a help in understanding the relative binding preferences of various cations. Most metal ions of biological significance are hard or intermediate between hard and soft. Most soft metal ions and soft ligands are poisonous and they interact with other soft species in the body. For Pb2+ the harder ligands are found in hemidirected structures and the softer ligands in holodirected complexes.Nature has devised many enzyme systems in which a metal ion interacts with the oxygen of a water molecule.If a water molecule can be dissociated into a hydrogen ion and a hydroxyl group, the latter can serve as a nucleophile in chemical a nd biochemical reactions.Nature has chosen activation of a water molecule as a means to obtain such a nucleophile in situation so that a chemical reaction can occur in a stereochemically controlled manner in the active site of the enzyme. The questions we ask are as follows: 1) how does nature ensure that the specific water molecule will be activated; 2) how does nature compensate for the lower water activation power of some cations over others (since a wide variety of metal ions may not be available in the particular active site and the enzyme has to do the best it can with what is available); and 3) how does nature ensure that the required reaction occurs. Ab initio molecular orbital and density functional calculations have been carried out to measure the extent to which a series of metal cations can, on binding with water, cause it to be dissociated into its component hydrogen ions (subsequently hydrated in solution) and hydroxyl ions. Initial data indicate that the charge of the metal ion plays a significant role in modifying the pKa of water. The binding enthalpies of a wide variety of metal ion monohydrates, M[H2O]2+ , have been published [21] but their deprotonation enthalpies are still under investigation. Geometry of Metal-Ion Binding to Functional Groups The geometries of metal ion-carboxylate interactions have been studied in order to determine the following: 1)which lone pair of an oxygen atom in a carboxylate group, syn or anti, is preferred for metal cation binding; 2) does the metal ion lie in the plane of the carboxylgroup; and 3) under what conditions do metal ions share both oxygen atoms of the carboxylate group equally? We found that cations generally lie in the plane of the carboxylate group . The exceptions to this mainly include the alkali metal cations and some alkaline earth cations; these metals ionize readily and form strong bases so it is not surprising that they have less specific binding modes. When the distance of the metal cation to the carboxylate oxygen atoms is on the order of 2.3-2.6 D, the metal ion tends to share both oxygen atoms equally. Otherwise one oxygen atom of the carboxylate group is bound to the metal ion and the other is not. Calcium ions often form bidentate interactions, while it is less common for the smaller magnesium ions. Imidazole groups in histidyl side chains of proteins bind metal ions in a variety of enzymes. One imidazole can, by virtue of its two nitrogen atoms, bind one or two metal ions, depending on its ionization state and the suitabilities of the metal ion. The bases in DNA can also bind metal ions. We have analyzed hydrogen bonding to and from nitrogen atoms in nitrogen-containing heterocycles for crystal structures in the Cambridge Structural Database. It was found that for hydrogen bonding, a slight out-of-plane deviation of the binding atom often occurs. Metal ions bind more rigidly in the plane of the imidazole group. The energetic cost of such deviations were analyzed by ab initio molecular orbital calculations. In an investigation of protein crystal structures in the Protein Databank it was found that the binding of metal ions to histidine in proteins is more rigid and the location of the metal ion is more directional. Thus, if an enzyme needs to control the location and orientation of a carboxylate or imidazole group, it can accomplish this better with a metal ion than by hydrogen bonding. Metal ions in proteins are often involved in structural motifs. When a metalloenzyme carries out its catalytic function it uses one of a few possible three-dimensional arrangements of functional groups around the metal ion to ensure the specificity of the required biochemical reaction. Thus, if such catalytic metal-binding motifs can be identified and categorized, then incipient reactivities of enzymes could be inferred from their three-dimensional structures. Such a categorization, however, requires an understanding of the underlying chemistry of any metal ion in the active site. One motif identified in the crystal structure of cobalt(II) formate consists of a carboxyl group in which one oxygen atom is bound to the metal ion and the other is bound to metal-bound water, to give a cyclic structure. This motif has been found in many metalloenzyme crystal structure , such as D-xylose isomerase . The roles of these motifs are of interest. The metal ion-hydrated-carboxylate motif (I) is planar and commonly found. It does not, however, affect the ability of the metal ion (in studies of Mg2+ complexes) to ionize water. On the other hand, for magnesium ions (which generally have a rigid octahedral arrangement of binding groups) it utilizes 2 of the 6 coordination positions and therefore serves to orient the arrangement of ligands, an effect we have labeled coordination clamping. Motif (II) is also found in several crystal structures such as that of the -subunit of integrin CR3 . It appears to help bind subunits together. A third motif (III) is found in D-xylose isomerase and involves two metal ions with several carboxylate ligands and a histidine ligand . The metal site that binds only oxygen atoms can bind substrate in place of the two water molecules and orient the substrate. The second metal ion site (with histidine as one ligand) then positions a metal ion-bound water molecule to attack the substrate. Roles of Metal Ions in Enzyme Action The crystal structure of mandelate racemase with bound p-iodomandelate provides a useful example of the importance of a metal ion in a reaction . The enzyme binds a magnesium ion by means of three carboxyl groups. The substrate mandelate has displaced water from the magnesium coordination sphere and binds by means of its carboxylate group and an a-hydroxy group.The magnesium ion will lie in the plane of the carboxyl group, as shown by our studies of metal ion-carboxylate interactions . The magnesium holds the substrate firmly in place so that the catalytic abstraction and addition of a hydrogen atom by His 297 or Lys 166 is precisely effected . The magnesium probably also aids this activity by affecting the electronic flow in the carboxylate and hydroxyl groups by mild polarization. We have found that metal ion coordination is better than a hydrogen bond in aligning a functional group; there is considerable flexibility in a hydrogen bond as we found for imidazoles . In the reaction c atalyzed by the enzyme mandelate racemase the magnesium ion binds substrate . A Histidine (His 297) and Lysine (Lys 168) are positioned to abstract a hydrogen ion from the substrate and, if it is added again from the other side, racemization occurs. Hydrogen bonding to a carboxylate group of the substrate helps to stabilize an enolate intermediate in the reaction. In catechol O-methyltransferase , a methyl group is transferred from the sulfur of Sadenosy[ methionine to catechol. The magnesium ion is oriented by a motif of type I and it binds substrate in such an orientation that a hydroxyl group is near the S-CH3 group, and the other hydroxyl group is held in place by a carboxylate group. There are many other examples of two-metal ion active sites, such as hemerythrin, alkaline phosphatase and superoxide dismutases (which have been well documented). These studies of the geometries and energetics of metal-ion ligand b inding can therefore aid in our understanding of metalloenzyme function Metals in the RNA worid By combining our limited knowledge of metal-ion-binding to contemporary RNAs and our more extensive knowledge of metal-ion-binding to proteins, it is possible to speculate on the role of metal ions in prebiotic molecular evolution. It seems clear that specifically bound metal ions coevolved with RNA molecules. Many of the mononuclear sites in Table 5 are formed with, or can be engineered into, small RNA fragments. Since such sites are highly hydrated and contain limited direct contact with the RNA, the observed affinities are only moderate, in the 1-1000 ÃŽÂ ¼M range. These sites are also expected to show limited specificity, predominantly dictated by the chemical nature of the ligands. Furthermore, in these examples, the RNA structures themselves are likely to be quite flexible and can accommodate a variety of metal ions with only minor distortions to the overall RNA fold. These minimalist sites are sufficient to stabilize the secondary and tertiary structures observed in these motifs. The metal ion sites generated on small RNAs appear to be capable of facilitating a variety of different types of chemistry. Activities range from the transesterification and hydrolytic reactions of small ribozymes (Pyle 1996; Sigurdsson et al. 1998) to the more exotic porphyrin metalation (Conn et al. 1996) and Diels-Alder condensation reactions (Tarasow et al. 1997) catalyzed by aptamers produced from in vitro selection experiments.These small RNAs have only limited amounts of structure and therefore are likely to position the catalytic metal ions by only a few points of contact. The relatively modest rate enhancements supported by catalytic RNAs such as these probably reflect the types of species that first evolved from random polymerization events. Very active metal ions might have assisted in this process but would have increased the danger of side reactions that would accidentally damage the catalyst. A striking difference between most RNA metal-binding sites studied thus far and those seen in proteins is the degree of hydration. Both structural and catalytic metal-ion-binding sites in proteins are predominantly dehydrated (Lippard and Berg 1995). Water molecules occasionally appear in the coordination spheres of these metal ions, but in these cases, they are often believed either to be displaced by the substrate when it enters the active site or to take part in the catalytic mechanism of the enzyme. Such protein sites also bind their metal ions much more tightly than the RNA systems. In fact, tight binding is a requirement for dehydrated sites, since there is a characteristic energy (ÄHhyd) associated with the hydration of any ion. The net binding energy upon coordination of the ion must account for the energetic cost of dehydration. The question arises, Why are such dehydrated sites not observed in RNAs? One possibility is that metal-binding sites in RNAs are intrinsically different from those in proteins. RNA has a much more limited set of ligands to use in generating a specific metal-binding pocket. Amino acid side chains containing thiols and thioethers are well suited to binding a variety of softer metals. In addition, the carboxylate side chains provide anionic ligands with great versatility in their potential modes of coordination. They can act as either terminal or bridging ligands and bind in either monodentate or bidentate geometries. The nucleotides, on the other hand, are much larger and more rigid than the corresponding amino acids. The anionic ligand in RNA, the nonbridging phosphate oxygen, is an integral component of the backbone and therefore is more limited in its conformational freedom than the aspartate and glutamate carboxylate groups. The heterocyclic ring nitrogens and the keto oxygens from the bases are held in rigidly planar orientations by the aromatic rings. This geometric constraint severely limits the ability of an RNA to compact encompass a metal ion and provide more than facial coordination and therefore complete dehydration. It also explains why the most specific metal-binding sites are not in the Watson-Crick base-paired regions of the structure where the conformation is too constrained. Instead, metalion- binding sites are clustered in regions of extensive distortion from the A-form RNA helices. There is also the question of the folding of RNAs relative to that of proteins. It is possible that in RNAs there is insufficient energy in the folding and metal-binding process to completely displace the waters of hydration around a metal ion. It has been suggested that in contemporary RNAs, modified nucleotides might be present to assist in metal ion binding (Agris 1996). A more straightforward possibility, however, is that most RNAs studied to date are structurally too simple. In these RNAs, most residues involved in metal ion binding are solvent-exposed. Thus, the RNAs have no real inside comparable to the hydrophobic core of a protein. The largest RNA crystallographically characterized to date is the P4-P6 domain. On the basis of that structure, it was proposed that an ionic core may substitute in RNA folding for the hydrophobic core of proteins such that the 3 ° structure assembles around a fixed number of discrete metal-binding sites (Cate et al. 1997). Even in this structur e, however, the most buried of the metal-binding sites are significantly hydrated. It could be that all metal-ion-binding sites in RNA are at least partially hydrated. One can imagine several advantages to using hydrated ions within the ionic core of a large RNA. Hydrated ions would span larger voids than dehydrated ions and allow looser packing of secondary structure elements. The hydrated ion also can accommodate a wide range of structural interactions through its orientation of the water molecules as compared to direct coordination of metal ions at every site. In addition, the energy associated with deforming the outer-sphere interactions should be significantly less than what would be observed for distorting the innersphere coordination. A consequence of RNAs having a core of hydrated ions is that one might expect this core to be much more dynamic than the hydrophobic core of a protein. In the modern protein world, metal cofactors are associated with a variety of reaction types, including electron transfer, redox chemistry, and hydrolysis reactions. Trans esterification and hydrolytic activities, however, are the primary catalytic behaviors observed in ribozymes. Did these other catalytic activities not develop until the dawn of the protein world, or are there undiscovered natural catalytic RNAs that are the ancestors of the early redox enzymes? Through the use of in vitro selection experiments, the scope of RNA catalysis has been significantly broadened is almost certainly capable of catalyzing these other classes of reactions, but it is still unclear whether there are naturally occurring examples. Such an enzyme would likely use a metal ion cofactor other than Mg(II), so the search for RNA molecules that naturally use alternative ions is of significant interest. A recent selection experiment showed that a single base change results in an altered metal ion specific ity for RNase P (Frank and Pace 1997). It is clear from this result that catalytic RNAs retain the ability to adapt to an everchanging environment, using the resources available to evolve and to overcome evolutionary pressures. Were RNAs to have evolved out of an environment devoid of metal ions, they probably would have found a way around the problems of folding and generating reactive functional groups. The primordial soup and all cellular environments that have evolved subsequently contained a variety of ions, however. Given the availability of metal ions, they will certainly play a significant role in the biology of current and future RNAs. Effect of metal ions on the kinetics of tyrosine oxidation by Tyrosinase The conversion of tyrosine into dopa [3-(3,4-dihydroxyphenyl)alanine] is the rate limiting step in the biosynthesis of melanins catalysed by tyrosinase. This hydroxylation reaction is characterized by a lag period, the extent of which depends on various parameters, notably the presence of a suitable hydrogen donor such as dopa or tetrahydropterin. We have now found that catalytic amounts of Fe2+ ions have the same effect as dopa in stimulating the tyrosine hydroxylase activity of the enzyme. Kinetic experiments showed that the shortening of the induction time depends on the concentration of the added metal and the nature of the buffer system used and is not suppressed by superoxide dismutase, catalase, formate or mannitol. Notably, Fe3+ ions showed only a small delaying effect on tyrosinase activity. Among the other metals which were tested, Zn2+, Co2+, Cd2+ and Ni2+ had no detectable influence, whereas Cu2+ and Mn2+ exhibited a marked inhibitory effect on the kinetics of tyrosine ox idation. These findings are discussed in the light of the commonly accepted mechanism of action of tyrosinase. Tyrosinase (monophenol,dihydroxyphenylalanine oxygen oxidoreductase; is a copper-containing enzyme responsible for melanogenesis in plants and animals, which catalyses both hydroxylation of tyrosine to dopa and its subsequent oxidation to dopaquinone (Hearing et al., 1980; Lerch, 1981). The first reaction, which represents the rate-limiting step in melanin biosynthesis (Lerner et al., 1949), is characterized by a lag period that has subsequently been explained in terms of a hysteretic process of the enzyme (Garcia Carmona et al., 1980). The extent of this induction time depends on various parameters including, besides pH and both substrate and enzyme concentration, the presence of a suitable hydrogen donor. Kinetic studies carried out on tyrosinases from various sources (Pomerantz, 1966; Pomerantz Murthy, 1974; Hearing Ekel, 1976; Prota et al Abbreviations used: dopa, 3-(3,4-dihydroxyphenyl)-alanine; SOD, superoxide dismutase. To whom correspondence and reprint requests should be addressed. 1981) have shown that dopa, in very low concentration, is the most effective reducing agent in eliminating the lag period, whereas other catechols, such as dopamine, adrenaline and noradrenaline, behave similarly to ascorbate and NADH and NADPH in only shortening it, even at high concentration. Tetrahydropterin, a well-known specific cofactor of other aromatic hydroxylases (Lerner et al., 1977; Marota Shiman, 1984), is also effective in stimulating tyrosinase activity, although to a lesser extent than dopa. At present, no other organic or inorganic substances have been reported to shorten or lengthen the lag period of tyrosine oxidation. Although metal ions are known to play a role in many biologi cal processes, little attention has been directed to their possible involvement in melanogenesis, particularly in the early enzymic stages .As a part of our continuing studies on the chemistry of melanin pigmentation (Prota, 1980; Sealey et al., 1982; Palumbo et al., 1983), we report the results of a survey on the effect of metal ions on the activity of purified Sepia tyrosinase, readily available in large amounts from the ink of the cephalopod Sepia officinalis thermostability of amalyse Three Metal Ions Participate in the Reaction Catalyzed by T5 Flap Endonuclease*à ¢- ¡ Protein nucleases and RNA enzymes depend on divalent metal ions to catalyze the rapid hydrolysis of phosphate diester linkages of nucleic acids during DNA replication, DNA repair, RNA processing, and RNA degradation. These enzymes are widely proposed to catalyze phosphate diester hydrolysis using a two-metal-ion mechanism. Yet, analyses of flap endonuclease (FEN) family members, which occur in all domains of life and act in DNA replication and repair, exemplify controversies regarding the classical two-metal-ion mechanism for phosphate diester hydrolysis. Whereas substrate-free structures of FENs identify two active site metal ions, their typical separation of>4 AËÅ ¡ appears incompatible with this mechanism. To clarify the roles played by FEN metal ions, we report here a detailed evaluation of the magnesium ion response of T5FEN. Kinetic investigations reveal that overall the T5FEN-catalyzed reaction requires at least three magnesium ions, implying that an additional metal ion is bound. The presence of at least two ions bound with differing affinity is required to catalyze phosphate diester hydrolysis. Analysis of the inhibition of reactions by calcium ions is consistent with a requirement for two viable cofactors (Mg2_ or Mn2_). The apparent substrate association constant is maximized by binding two magnesium ions. This may reflect a metal dependent unpairing of duplex substrate required to position the scissile phosphate in contact with metal ion(s). The combined results suggest that T5FEN primarily uses a two-metal-ion mechanism for chemical catalysis, but that its overall metallobiochemistry is more complex and requires three ions. Key cellular processes such as DNA replication, DNA repair, RNA processing, and RNA degradation require the rapid hydrolysis of the phosphate diester linkages of nucleic acids. The uncatalyzed hydrolysis of phosphate diesters under biological conditions is an extremely slow process with an estimated half-life of 30 million years at 25  °C (1). Protein nucleases and RNA enzymes produce rate enhancements of 1015-1017 to allow this reaction to proceed on a biologically useful time scale. Most enzymes catalyzing phosphate diester bond hydrolysis have a requirement for divalent metal ions. Based largely upon crystallographic observations, most metallonucleases are proposed to catalyze reactions using a two-metal-ion mechanism (Fig. 1a) analogous to that suggested for the phosphate monoesterase alkaline phosphatase (2, 3), although this view is not universally accepted. Three recent reviews present contrasting views on the roles of metal ions in protein nuclease and RNA enzyme reactions and illustrate this controversy (4-6). One family of metallonucleases over which there has been considerable mechanistic debate are the flap endonucleases (FENs)3 (7-12), which are present in all domains of life and play a key role in DNA replication and repair. Unlike most metallonucleases, which typically possess a cluster of three or four active site carboxylates, the FEN active site is constructed from seven or eight acidic residues located in similar positions in FENs from a range of organisms (Fig. 1b, see also supplemental Fig. S1) (7, 9, 10, 13-16). Several FEN x-ray structures also contain two active site carboxylate-liganded divalent metal ions, designated as metals 1 and 2 (9, 13-15). The position of metal 1 is similar in all cases, but the metal 2 location varies. In all but on