Ei-lchiro Ochiai The University of British Columbia Vancouver. B.C., Canada
Principles in Bioinorganic Chemistry Basic inorganic exercises
The biochemical roles played by essential inorganic elements and compounds are 1 ) structural, 2) carryingand transportingelectrons and oxygen, 3) catalytic roles in oxidation-reduction reactions, and 4 ) catalytic roles in acid-base and other reactions. Many inorganic elements and their compounds are now known to he essential to organisms ( I ) . Organic compounds are of course essential, because they provide organisms with such essential compounds as proteins, nucleotides, carbohydrates, vitamins, and so forth. Inorganic compounds, particularly metallic ions and complexes, are essential cofactors in a variety of enzymes and proteins. They conceivably provide essential services which cannot he or can only poorly he rendered by organic compounds. The roles played by essential inorganic elements and compounds are (1) structural (2) carrying and transporting electrons and oxygen, (3) catalytic roles in oxidation-reduction (including oxygenation) reactions and (4) catalytic roles in acid-base and other reactions. The type of question we are interested in here is why a certain inorganic element (compound) is specifically required for a certain function in hiological systems. For example, why is cobalt uniquely required for vitamin BIZand the enzymatic reactions dependent on BIZ? Why not iron or copper? We think that questions of this type may he answered a t least partially in terms of basic inorganic chemistry. This is what this article attempts to show. First let us lay out some basic principles regarding the above question. They are 1) Rule of abundance 2) Rule of efficiency 3) Rule of basic fitness 4) Evolutionary improvement of efficiency and specificity
We will illustrate these principles by a few examples. Rule of Abundance The rule of abundance may he stated as: When a function can he accomplished by two or more entities, organisms would utilize the more ahundant, readily availahle one. Examples of this rule are numerous. As a general rule, the lighter elements are more ahundant. The elements essential to organisms are mostly light elements, lighter than atomic number 34 (selenium), with exceptions of iodine and molyhdenum. Molybdenum, as Mo042-, is present in arather high concentration, as high as iron in sea water. The concentration of zinc in sea water is also on the same order of magnitude as that of iron; the copper concentration is about half as high. These four elements are the most frequently found in the catalytic sites of enzymes. The use of the most ahundant alkali metals, sodium and potassium, in controlling ion balance and enzyme activities is also in accord with this rule. Most organisms utilize calcium compounds such as carbonate and phosphate as protective and skeletal material. Undoubtedly this is due to the insolubility of calcium carbonate and phosphate. However, the corresponding strontium compounds are equally insoluble and could substitute calcium compounds. It is obvious that calcium is much more ahundant than strontium (1,Z). An interesting exception is a group of marine protist, radioralian; their outer skeletons are made of either strontium sulfate or silica. Somehow they could not
develop a mechanism to utilize more ahundant calcium or their specific need may not he satisfied by calcium compounds. Zinc(I1) in zinc-enzymes can he in most cases replaced by cohalt(I1) in uitro without losing catalytic activity (3).If organisms are grown in cobalt-rich media, they can produce enzvmes in which Zn(I1) is renlaced bv Co(I1). For examole. . . in thepresence of relatiwly whrn E w h ~ n c h i ocoli is active6"CdI 1 ) hieh concentration ofFUCo~II~.a~nt:~l~ti~aIIv alcaline phosphatase is obtained (4): ~ h u izinc , and cobalt seem to he interchaneeahle, hut oreanisms selected zinc hecause zinc is much more ahundant both in sea and earth's unner .. crust. A general discussion of the relationship between the abundance of elements and their essentialitv or toxicitv was made elsewhere (1,Z).
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Rule of Efficiency The rule of efficiency asserts that organisms would choose the more efficient entity as long as i t is readily availahle. Flavcdoxins (5)and ferredoxins function as electron-carriers in very similar ways, being interchangeable in most cases. However. their comoositions are entirelv different. Flavodoxin contains'flavin mkonucleotide (FMN) as the prosthetic group, whereas the functional units in ferredoxins are ironsulfur complexes. Flavodoxin in general is less efficient than ferredoxin. and the svnthesis of flavodoxin occurs onlv durina growth in & ~ n - ~ omedia o r in a number of microorganisms. FO; exam~le.in the case of Pe~tostre~tococcus elsdenii, iron-rich crlls were iound not tocontain flavodoxin and iron-deiiriencies brought ahuut itsdr nouo synthesis (51. Rule of Basic Fitness Primitive (in historical sense) organisms tried to improve their survival chances by tapping whatever resources were available to them. For example, they might have explored manv transition metal comolexes in order to effect the oxidation-reduction reactions.-ASthe results of this endeavor, thev selected iron. comer. and molvhdenum and their comple&esto effect their desiied oxidation-reduction reactions. This third basic principle might he stated as: an inorganic element (generally a metal) to be selected should have a basic ability or potential to carry out the desired function. That is, a certain element (or elements) would inherently fit to a particular function. As mentioned earlier, most organisms utilize calcium compounds as protective material. An important group of organisms, diatom, use silica as their outer cover, despite the fact that silicon is much less ahundant than calcium and strontium in sea water. Diatom is a kind of algae. There are a vast number of different kinds of algae and all algae other than diatom do not have anv cover of inoreanic material. though there are a few greenand red algae, mostly extinct; which have sliehtlv calcified walls. Somehow the oredecessor of diatom happened to try a coat of silica and f&d it very useful for its survival. Perhaps some other algae might have Volume 55, Number 10, October 1978 1 631
F gum 1 The rw~ctlonpotentla r of some mpmam metal Ions and omer redox systems 0 represents me red~ctlanpotentla1 of a metal ,007 m an aqueous medium either at pH = 0 or at pH = 14. or the reduction potential of a simple metal complex. See the text for a more detailed explanation.
tried limy coats but did not find them useful. Probably it is due to the fact that silica forms transparent material whereas limv coats are often much less transnarent. Aleae. of course. have to absorb sunlight to support h e i r phot'ynthesis. An element may have ranges of capacities modulated by external factors such as coordinating ligands. However, these ranges are not limitless, and are more or less confined within certain values, particularly when the kinds of external factors are limited. Some of the important restrictions in the case of ordinary biological systems are: (1) the medium is water, (2) the range of temperature is rather narrow, and (3) the possible coordinating ligands are limited; they are proteins, carhohydrates. nucleotides.. lipids, such . and a few other specific ligands . as porphyrins. The catalytic effects of a metal ion can be characterized by many factors (6). They are: (1)continuous parameters such as reduction potential and Lewis acidity and (2) discontinuous (discrete, parameters such as numher of valence denrons and favorable coordination number (structure,. The parameters in category (1)are modulated by ligands and other external factors such as pH of the medium, hut those in category (2) are much less influenced by ligands and others. Let us take reduction potential as an example and illustrate how the rule of basic fitness may work. Figure 1shows the reductiou potentials for several important metal ions and other redox systems. The dotted lines represent the oxidation and reduction of water and the pH-dependency of the reduction ootentials. The points shown bv 0 are the reduction poteuti&s of the aquo species a t p H = d a n d those of the corres~ondinehvdroxo complexes a t pH = 14. The two points a t p~~ and i 4 are joined b i a straight line which doesnot necessarilv represent the DH dependence of the reduction potential: ~ h reduction k pkenti& for a few ordinary compl&es are shown on the extended broken lines. T h e reductiou potentials (at p H 7) of the metalloenzymes and metalloprotiins containing copper and iron are shown by 0 or A on the respective straight lines or the broken lines in Figure 2. The following items are some of the points that we can infer from Figures 1and 2. (1) Since the species whose reduction potentials lie outside of the
decomposition lines of water (dotted lines) would (in thermo632 1 Journal of Chemical Education
Figure 2. The redunion potentials of iron- and copper-enzymes and proteins at pH 7. me point represents merely the reduction potemial value: the pasition on the pH axls has no meaning. See Figure 1 for the explanation of the dotted lines and the shalght solid or broken lines.
dynamic sense) either oxidize or reduce water, they may not he suitahle for a catalytic entity working in aqueous media. This consideration alone would probably exclude Co(III)/Co(II), Sn(IV)/Sn(II), and Cr(III)/Cr(II) systems as candidates for redox catalysts (enzymes). 2) The entire range of the reduction potentials of a variety of iron-enzymes and proteins is confined in the potential range of the iron-aquo system. The potentials of iran-sulfur proteins are closer to that of the Fe21"S&2FenS system. These facts may indicate that the prehiotic catalysts for those reactions carried out today by iron-containing enzymes and proteins might have been Fe(H20)6..(0H). and FeS/FezSa or their simple complexes. These simple compounds were later incorporated into porphyrins and proteins, leading to the formation of mare efficientand selective catalysts, the potentials, however, did not change very widely. In other words, these simple compounds that might have been readily available to precursors of organisms or primitive organisms are basically fit to the specified requirements (chemical reactions). It should he noted, however, that iron porphyrins themselves might have been the first catalysts,since porphyrins are known to be one of the mast ancient compounds synthesized abiotically (7). 3) The situation of copper species is slightly different from that of iron. Cuprous aquo species are not stable and disproportionate into Cu(0) and Cu(I1).This would preclude the use of the simple aquo Cu(II)/Cu(I)system as an axidation-reduction catalyst. Instead, such simple complexes as Cu"C12/CuClz-, C~~(pyridine)~Cu'(p~ridine)~, or similar complexes could have provided the basis for prehiotic or primitive catalyst. Cu(I1)-C1species could indeed he predominant in a marine environment. The reduction potentials of these compounds are basically fit to those of present day copper enzyme and proteins. 4) The reduction potentials of 02-/H202 and 02/02- are +0.96 V and -0.45 V (at pH I ) , respectively. Suppose that the mechanism of superoxide dismutase reaction is simply as follows
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0% + M"+ + 2H+ H202+ Mn+l+ M"+'+ + 02- M"++ o2 Then it would he inferred that aredox system whose potential lies somewhere in the middle of the range +0.96 V to -0.45 V would function as a catalyst for the superoxide dismutase reaction. Figure 1indicates that such systems are Fe(III)IFe(II), Cu(II)/Cu(I),and Mn(III)/Mn(II). In fact, superoxide dismutases from different sources contain copper, iron, or manganese
(8). A superoxide dismutase containing copper was found to have a reduction potential of t 0 . 4 2 V (9).As far as the reduction potential is concerned,the aquo V(IVVV(II1)system is also fit far superoxide dimutase function. One of the functions of hemavanadin, which is a vanadium-containing protein found in a marine invertebrate, ascidian, might well be a superoxide dismutase.
Let us turn to another example, oxygen-carrying proteins (10). This function can he translated into the reversibility of the reaction to bind oxygen; that is M"+ t O2+ [Mn+'+02-1: AG, 2M"+ t O2+ [Mn+1+022-Mnt1+]: AGb In order for these reactions to be reversible, AG. or AGb should be roughly -2 to -12 kcallmole (9). If AG, (or AGb) < -2 kcallmole, the forward reaction occurs to only a small extent. On the other hand. if AG.- (AGA) . -. < -13 kcallmole. the reaction goes essentially to completion and the reverse reaction occurs to a neelieihle extent. A rouah estimate for AG, and AGa was madifr& reduction potentials and is given in the table (10). This table indicates: (1)the reversible oxyRough Estimates of AG. (kcallmole) and AGb (kcallmole) M*
Ti(ll)aq
Ti(ll1) (in 5 FthPOd Waq Cr(ll)aq Mn(l1) (in 8 FH2S03 Mn(ll)/CNFe(l1)(pH 5 2) Fe(l1)(pH 6)
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Fe(OHl2 Fe(ll)/CNC0Ill)aq Co(lllNH3 Co(ll)/CN-
Cull)/aq Cu(I)/NH,
AG .
AGb
t1.7 t6.9 t4.4 t0.9 +47.7 t5.2 t26.0 tl0.0 -2.6 +16.6 +52.7 t12.6 -9.0 +13.8 +10.1
-29.2 -19.0 -24.0 -31.0 t62.6 -22.4 t23.2 -11.8 -38.0 -4.4 t72.6 -7.6 -50.8 -4.8 -12.6
genation of type (a) is very probable with some Fe(I1) and COW)complexes and possibly some Cr(I1) complexes, and (2) the reversible oxygenation of type (b) may be carried out by some Fe(II), ColII), and Cu(1) complexes. This conclusion fits the known facts (9). An example uf the efferr of discrete parameters seems to be vitnmln RI,, cobalamin. The merhanisms of R I roen~ zyme-dependent en~ymaricrenctinnsarestill under intensive studv and are far from settled (11. 12). However. the imvortant ;eqnirements tcm the cilndidates for the job appear to he rather wrll dt+ined. Thw are: (11it should readilv take three consecutive oxidation states, for example, I, If and I11 in aqueous media, (2) the lowest oxidation state should be highly nucleophilic, and (3) the middle oxidation state should perhaps have one unpaired electron (11). The requirement (2) implies that the lowest oxidation state of the catalytic metal ion should have d@or d'O configuration. This condition may be satisfied by Fe(O), Co(I), Ni(O), and Cu(1). Fe(O), and Ni(0) are not readily attainable, particularly in aqueous media. This leaves us Co(1) and Cu(1). Cu(1)is not particularly nucleophilic and Cu(II1) is not readily obtained. Only a Co(III)/Co(II)/Co(I) system satisfies all these requirements. This explains why cobalt (cobalamin) uniquely fits the job (11). , . Other applications of the rule of basic fitness to more specific cases will be discussed elsewhere. Evolulionary Improvement of Efflclency and Specificity The rule of basic fitness dictates which element(s1 is suitable for a specific enzymatic reaction only in terms of thermodvnamics. The element or its simpler com~oundsselected maybe able to do the required function but may not he very
efficient in the senses of kinetics and specificity. The substrate svecificitv and the efficiencv seem to be controlled mainly bv the prot~-inportions of meialloproteins or metalloenzy& Thus, the improvement of these functions must have m k m place in early stages oie\.oluriun, involving rhe mutationi in the genetic rodes. We know little ahour this, hut we may he able-to rationalize some of the results from an inorganic standpoint. What we are concerned with here is the efficiency of many enzymes. Their rates are usually very much higher than those of simple model compounds. Vallee and Williams (13) proposed that the structure of the active site of an euzyme, particularly a metalloenzyme, is rather distorted or strained and that it is responsible for its high catalytic activity. They called it "Entatic Effect!'Let us now translate this into chemical language. Very generally speaking, the rate is most dependent on the activation enerev. The activation enerev can be defined in terms of ahsolu&"rate theory as the pote&d energy difference between the initial and the transition state. that is. the activation energy AG*, for the process ML S= (MLS)*, where ML is a metal comolex and S is a substrate includiia electron. A significant p&,n of I\(;* comes from the enerknere%sary to rrurganize the structure of MI. sn that MI. take the s t r w ture i l l (MIS)'. 'I'hereiore, the rloser the structurr nl' t h ~ initial state MI. is tothat of the transition state, rhe lesser X* becomes. A few examples will suffice to illustrate this puint. Blue copper proteins (14) surh ax plastocynnin and amrin funrtim as elrctron-carriers, in which wpper oicillnlrs Iletween Cu(l1)and Cu(l).The fmvrahle roordinatinn structure uf C'utll) is square planar and thnt of ( U l J is wtrahedral.'l'he redurtiun of a squnre planar Cu(II),therefore, wollld rvquire a slgnilicant rearranxement in the structure ahi~ufcr,pper ion. One way toreduce thiscost is tostart with a wmpo~lndwhnw structure is hetween the regular square planar and tht. rryuli~r terrahedral one. There is now rtmvint:ine evidence that the rourdinntinn structure ahnut cnppcr atom in the hlue proteins I. 15) for review). is inderd ~listc~rtcd-tetrahedral (see . Other examples are zinc enzymes (16). The structural studies by X-ray crystallography and by spectroscopy of the cobalt (11)-substituted enzymes have established that the coordination structures about zinc atom in carbonic anhvdrase, rarhoxyprptidase A, and alkaline phosphatase are distortrd tctrnhedra, beine between a rerular tetrahedron and a five-coordinate trigond bipyamid ( 3 ) . ~ h function e of these enzymes is to hydrate the substrates. Not only the substrate but also the water molecule would have to be activated. The distorted tetrahedral structures of the enzymes usually have one water molecule and three amino acid residues coordinated about the zinc ion. If the coordination structure about the zinc atom is distorted from tetrahedron in such a way that the substrate can readily approach the zinc ion (forming a fivecoordinate bipyramidal type of structure), the whole reaction would be facilitated. In a regular tetrahedron, this binding of an additional ligand would not be very easy.
+
Literature Cited (11 Ochisi. E. -I., "Bioinorganic Chemistry: An Introduction:
..... ,a,,
A l l p and Bacon. Inc.,
(21 Ochisi, E. -I.,J. CHEM. EOUC., 51,235 (1974): Chap. 1and 17 in ret ( I ) . (31 0chiai.E. -1,Chsp. 13in ref. (11. (4) Harri3.M. I.,andColeman, J. E.J.B i d Cham.. 244,709 (19641. (51 Mayhew. S. G., and Ludwig, M. L.. in "The Enzymes, 3rd Ed.. XiI." (Editor: Boycr, P. 0.1Academic Press. 1975, p. 57. (61 Ochiai, E. -1, Cooidin Chem. Rev., 3,49(19681. 17) a,&, Miller. S. L., and Owel, L. E.."TheOrigins oiLih on Eeuth."Pmntice-Hall, Inc.
1974.
181 Fee, J. A , and di Corleto, P. Q., Bioch~misiry,12,4893 (1973). (91 Fridovieh. I.. in "Free Radicals in Biolagi." Val. 1,(Editor: Pryor, W. A,), Academic
Press. 1976, p. 239. (10) Ochiai. E. -I., J. Inarg, Nucl Chem., 35, 3375 (1973): Chap. 10 in ref. (11. (11) Ochisi, E. -I.. J.Inm. Nuei. Cl&rm.,31.351 (1975):Chap:lZinrei. ( I ) . (12) Able., R. H., in "Bialogicsl Aspem of Inorganic Chemistry," (Editors: Addison. A. W., ef 81.1, Wiiey-Interscience, 1977, p. 245. (13) Vallep,B. L.,snd Willisms,R. J. P..Proc.NoI. Aced Sci. (US), 59.498 (19681. (14) Fee, J. A.,Slrvel.andBondg., 23.1(19751. (15) Yokoi, H..andAddison,A.W..lnorg.Chem., 16,1341(19771. (16) Chlebmuski. J. F., and Coleman, J. E., in "Metal Ions in Biological Syatcms: Val. 6. (Editor Sigel. H.1,MsreelDekker. 1976,~.1.
Volume 55. Number 10, October 1978 1 633
