On the Nature of Boundary-Organized

Biomineralization (BOB)

Stephen Mann

School of Chemistry, University of Bath

INTRODUCTION

The biological regulation of structural inorganic chemistry gives rise to the

phenomenon of biomineralization . In this process inorganic elements are extracted

from the environment and selectively precipitated within biological space for

functional use . A range of bioinorganic solids can be formed which can be classified

according to the degree of biological control over their crystallochemical properties .

Two extreme regimes are possible . Firstly, "biologically induced" mineralization

involves precipitation of inorganic solids in the absence of any active biochemical

mediation . Mineralization occurs through secondary events involving metabolic end

products such as C02, H+, NH3, and ions in the surrounding extracellularenvironment. A wide range of solids can be formed because both cations and anions

involved in these precipitation reactions can vary extensively according to the local

environmental conditions . In contrast, "biologically controlled" processes of mineral-

ization involve the active biological regulation of inorganic solid state reactions and

therefore include only a limited number of cations and anions due to restrictions in the

abundance and bioavailability of elements in the surrounding environment [1] . Table 1

lists the major biominerals known to be formed under biological control .

In this paper I outline some of the ideas and results that have been generated during

my postgraduate and postdoctoral association with Bob Williams at Oxford (1978-

1984) . We have been concerned with biologically controlled mechanisms of

biomineralization and have adopted structure-determination techniques such as high-

resolution transmission electron microscopy (HRTEM) to aid the elucidation of these

processes . Our work has highlighted the precision of control inherent in bioinorganic

Address reprint request to Dr . S . Mann, School of Chemistry, Universtiy of Bath, Bath BA2 7AY U .K .

Journal of Inorganic Biochemistry 28, 363-371 (1986)

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© 1986 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave ., New York, NY 10017

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364 S. Mann

CONTROLS IN BIOMINERALIZATION

solid state reactions that manifests in the formation of mineral phases withpredetermined sizes, morphologies, structures, crystallographic orientations, andfabrics (microscopic particulate arrangements) . Note that these properties are ofcritical importance in the systematic and reproducible synthesis of inorganic solids forselective functional use in industrial applications such as catalysis, electronic andmagnetic storage devices, and ceramic-based products .

A major feature of our work has focused on the ideas of BOB (boundary-organizedbiomineralization) . The BOB concept, in principle, is simple yet far-reaching, andenables a rationalization of many complex processes that have otherwise been subjectto much heated debate . In essence, the concept states that the controlled formation ofbioinorganic solids arise from the local involvement of organic boundaries, such aslipid, protein, and polysaccharide surfaces, within the mineralization zone . As will bedescribed below, this involvement provides (i) spatial localization of precipitationreactions ; (ii) spatial constraints on mineral development, (iii) chemically specificreaction environments, and (iv) stereospecific surfaces for nucleation and growth . Iwill first describe some general characteristics of biologically controlled mineraliza-tion .

The central problem in attempting to understand biomineralization processes arisesfrom the difficulty in placing such processes in the context of the overall celldynamics . We can begin by identifying control at several different levels (Fig . 1) . Wenote that the physicochemical events (solubility, supersaturation, nucleation, andgrowth) that lead to mineralization are determined by a hierarchy of control levelsinvolving biochemical, bioenergetic, and ultimately, genetic factors . Only when wecan begin to understand the interconnections between these different levels oforganization within the cell will we be able to place biomineralization in a more

TABLE 1 . Biologically Controlled Inorganic Solids

Formula Mineral Function

CaCO, Calcite ExoskeletonsAragonite Gravity devicesV ateri to

CaJPOS), (OH), HydroxyapatiteBrushite Structural supportAmorphous Ca stores

MSO4 Gypsum Gravity devices(M = Ca, Ba, Sr) Barite Structural support

Celestite

Sift Amorphous ExoskeletonsDeterrence

Fe-oxides Magnetite MagnetoreceptionGoethite Structural support (teeth)Lepidocrocite Fe storage and transportFerrihvdrite

BOUNDARY-ORGANIZED BIOMINERALIZATION 365

GENE POOL

BIOENERGETICSV

BIOCHEMICAL

*ENVIRONMENT=

FIGURE 1 . Diagrammatic representation of control processes in biomineralization . There areseveral levels of regulation that interconnect within the living organism . Interaction between thephysicochemical and biochemical domains is mediated by spatial boundaries .

detailed perspective . However, we have gained much information concerning thebiochemical-physicochemical interface through our recent structural and analyticalstudies (see Refs . 3-9 for examples) .

CHARACTERISTICS OF BOB (BOUNDARY-ORGANIZEDBIOMINERALIZATION)

Reference to Figure 1 indicates that the interaction between the physicochemical andbiochemical domains in biomineralization processes is mediated by spatial boundaries .The chemical, stereo, and structural properties of these boundaries will be criticalfactors in the control and organization of bioinorganic solid formation . We will nowdiscuss these characteristics in more detail .

Spatial Localization

Figure 2 shows a generalized scheme for biomineralization [2] . Mineralization canoccur at intracellular sites defined by, for example, enclosed lipid boundaries in theform of vesicles . Alternatively, extracellular mineralization can occur within spatialboundaries established by extracellular matrices in the form of extended organicpolymeric frameworks . In both cases there is close, and often dynamic, associationbetween the development of inorganic and organic phases . BOB thus provides aneffective center for localized growth . Note that vesicles can act as transport devices for

3 66 S. Mann

FIGURE 2 .details .

ACCELERATORS j

AND INHIBITORS'

/ EXTRACELLULAR ORGANIC MATRIX

Generalized scheme for biologically controlled biomineralization . See text for

ions or bioinorganic solids resulting in translocation of these centers to functional siteswithin the organism . Other specialized components, such as low molecular weightorganic compounds, can act as accelerators and inhibitors in both intra- andextracellular biomineralization .

Various biological molecules can be assembled to provide spatial localization . Inparticular, bilayer lipid vesicles have been implicated in many biomineralizationreactions . These vesicles may be formed in the Golgi apparatus or derived from theinvagination of cellular membranes . The former are likely to contain polymer matrixproteins, whereas the latter are often sites of high acidity (for example, lysozomes) . Inmany cases the primary vesicles are fused to provide larger enclosed volumes . It isoften unclear whether mineralization occurs concurrent with or before or after theseaggregation events .

Alternatively, protein vesicles can provide the necessary spatial localization forbiomineralization . For example, the iron storage protein ferritin is constructed with anunique molecular microarchitecture in which 24 protein subunits are assembled into ahollow sphere with an internal diameter of 80 A . Iron(III) oxide deposition can thenoccur under controlled conditions within the cavity delineated by the assembledprotein .

Spatial organic nets, in which biological macromolecules are intertwined ingeometric arrays, can also serve as organized boundaries for biomineralizationreactions . These frameworks are usually extracellular and provide the functionalorganic matrices for bulk mineralized structures such as in bone, teeth, and shells . Therigid structure of these spatial nets is formed by strands of connective tissue such aslong multiple helices of protein, for example, the glycine-proline-alanine-richcollagen protein in bone . Invertebrates, in contrast, often utilize protein-chitincomposites in the form of spatially organized fibrils as organic matrices in

BOUNDARY-ORGANIZED BIOMINERALIZATION 367

biomineralized structures such as limpet teeth . Alternatively (3-pleated sheet proteinscan provide rigid boundaries for calcification reactions in invertebrate shells . In thesesystems the organic framework is assembled into lamellae such that individualaragonite crystals grow between adjacent layers resulting in a "brick-and-mortar"shell wall arrangement .

Spatial Constraints

An immediate corollary of providing a discrete locale for mineralization is theconsequent spatial constraint placed on the extent and direction of mineral develop-ment. In consequence, bioinorganic solids can be regulated in both size andmorphology by organized boundaries . BOB thus directs development towards amaximization in functional potential .

We have investigated several systems, both biological [3-9] and chemical [10-12],involving the use of spatial constraints in precipitation reactions . A fascinatingbiomineralization system we have studied is the formation of magnetite (Fe 3O4)crystals in magnetotactic bacteria [4, 5] . In these organisms the size of each crystal isprecisely determined by a surrounding organic envelope . All the crystals studied todate have dimensions that fall within the magnetic single domain size range (50-100nm), thus optimizing the magnetic moment of each particle and hence thecorresponding response of the cell to the geomagnetic field . (Particles smaller than thisrange would be superparamagnetic ; those above would comprise magnetic domainwall boundaries . Both cases result in a reduction in the bulk magnetization .)

Regulation over biomineral shape can be exerted through the biological design ofvesicle geometry . More energetically demanding geometries are possible ; forexample, the formation of curved 3-µm silica rods in certain marine protozoa isdirected within preformed curved elongated vesicles (Fig . 3) [13] . Similarly thegeometric patterning of diatom silica frustules [14] and calcified plates of coccoliths[15] occur within preformed stereospecific vesicles . In general, vesicle patterningappears to be determined by underlying cytoskeletal elements in the form of proteinmicrotubules .

In a related way, constraints are placed on the growth of bioinorganic solids withinspatial nets . Goethite single crystals, mineralized within the protein-chitin matrix oflimpet teeth, show remarkable growth distortions along the [001] direction (Fig . 4),reflecting spatial distortions in the apposed organic boundary [4] .

Localized Chemical Control

The controlled nucleation and growth of bioinorganic solids involves the temporal andspatial regulation of chemical processes . The essential physicochemical property to becontrolled is the level of supersaturation within the mineralization zone . This factorrepresents the nonequilibrium status of the mineralization environment and hasfundamental influence over both nucleation and growth pathways . In consequence, thestructure and morphology of biominerals will be highly sensitive to changes insupersaturation .

The precision of replication of biomineralized structures requires the organizationof reaction volumes in which there is a high degree of control over localizedsupersaturation levels . The structural, compositional, and electrochemical nature oforganic boundaries will be paramount in achieving this specificity . Several mecha-

368 S. Mann

FIGURE 3 . Section through a cell of the choanoflagellateS.diplocostata Ellis showing an intracellular curved silica rodformed within a preformed curved vesicle (arrow) adjacent to theendoplasmic reticulum (ER) [13] . Bar represents 500 nm .

nisms are possible (Fig . 5) . Firstly, the incorporation of selective ion pumps and othergated systems into organic membranes provides a mechanism by which differentialtransport of ions and molecules into the mineralization zone can be attained . Note thatin the ferritin molecule it is the polarity and size of the channels between proteinsubunit dimers that generate this molecular discrimination . In general, energization ofmembrane-bound pumps provides facilitated transport against electrochemical gradi-ents, which results in increases in localized supersaturation . Another possibility is theinvolvement of redox processes and complexation/decomplexation reactions forcations such as Fee' /Fe'' and Call, respectively . Both these mechanisms can againfacilitate ion transport against chemical or electrical gradients . Anion concentrationprofiles often involve the mediation of enzymes such as carbonic anhydrase (forHC03) and alkaline phosphatase (for HP0 4 1- ) . Changes in both ionic strength andpH by the differential pumping of ions not directly involved in the precipitationreactions may also influence the level of localized supersaturation via changes in ionactivities and dissociation constants . Finally, water movement across the organizedboundary may also be important. Clearly, BOB generates the appropriate chemistryfor subsequent growth .

The transport properties of the organized boundary will also play an important rcim,in assisting the progress of mineralization since ions required for further growth willbe continually supplied (at influx rates that may critically affect the crystallochemical

BOUNDARY-ORGANIZED BIOMINERALIZATION 369

FIGURE 4 . Pseudoacidular single crystal of biogenic goethite (a-FeOOH) showing markedgrowth distortions (arrows) caused by spatial constraints in the organic matrix of limpet teeth .Bar represents 200 run .

properties) and ions and molecules produced in the precipitations reactions continuallyremoved from the localized reaction volume . For example, iron oxide mineralizationgenerates H+ , whereas silicification produces H2O as a by-product of the condensationreaction . Mineralization of the biomineral can also be assisted by changes in thereaction mechanisms involved in growth through the selective incorporation within thelocalized volume of organic molecules that act as inhibitors, or mediators . In general,these processes of reaction-assisted growth (RAG) and transport-assisted growth(TAG) of BOB are tailored to some functional value within the organism .

Stereospecific Interfaces

An organized boundary in biomineralization provides an interface at which chemicalinteractions may occur . The possibility of ion binding will have important repercus-sions on nucleation since the surface energy required to form the new (mineral)interface can be considerably lowered . It has recently been proposed [16] that thecluster of acidic amino acid residues at the subunit dimer interface along the interior ofthe ferritin molecule could act in this way, providing a low-energy zone for iron oxidenucleation .

An extension of this idea leads to the concept of organic-matrix mediated epitaxy asinvoked, for example, in the molluscan calcareous shell [17] . In this system there is aclose correspondence between the lattice spacings in the ab plane of aragonite andthose in the underlying 13-pleated sheet protein . The presence of aspartate side chains

370 S. Mann

CONCLUSIONS

REFERENCES

FIGURE 5 . Possible mechanisms for controlling localized supersaturation in biomineraliza-tion processes . See text for details (MC = cation complex, E,, E, = enzymes, MX =biomineral) .

at regular repeat distances along the protein backbone then presents a negativelycharged net of correct periodicity to match that of Ca 2+ in the ab plane . This one-to-one correspondence consequently not only lowers the activation energy for nucleationbut generates a precise growth orientation (viz ., perpendicular to the ab plane) on thedeveloping mineral as experimentably observed. Although epitaxial relationshipsbetween inorganic minerals are well known, no verification for the interactionbetween inorganic minerals and organic substrates has as yet been presented in modelsystems . Hence the precise biological mechanism of these interactions is open tofurther exploration . However, it seems clear that BOB generates special conditions forthe initiation of growth and direction of development .

This paper has attempted to illustrate the importance of BOB (boundary-organizedbiomineralization) in aiding our understanding of biological systems . Inorganic solidstate processes are of extreme importance in biology and require much further study .The field is a new horizon for bioinorganic chemists, At present we owe much of ourchemical understanding and insight to Bob Williams at Oxford, whose intellect andcontinual enjoyment for science has provided the inspiration for the work and ideaspresented in this paper .

1 . R . J . P . Williams, Proc. R . Soc. London B 312, 361 (1981) .2 . S . Mann, Struct. Bond. 54, 125 (1983) .3 . S . Mann, J . V. Bannister, and R . J. P . Williams, J. Mol. BioL 188, 225 (1986) .4. S . Mann, C . C . Perry, J . Webb, and R . J . P . Williams, Proc. R . Soc. London B 227,

179 (1986) .

BOUNDARY-ORGANIZED BIOMINERALIZATION 371

5 . S . Mann, T . T . Moench, and R. J . P . Williams, Proc. R . Soc. London B 221, 385(1984) .

6. S. Mann, R. B . Frankel, and R . P. Blakemore, Nature 310, 405 (1984) .7 . C . C . Perry, S. Mann, and R . J . P . Williams, Proc. R. Soc. London B 222, 427

(1984) .8 . S . Mann, S . B . Parker, M . D . Ross, A . J . Skarnulis, and R . J . P. Williams, Proc. R .

Soc. London B 218, 415 (1983) .9 . S . Mann and R. J . P. Williams, Proc. R . Soc. London B 216, 137 (1982) .10 . S . Mann, A . J . Skarnulis, and R . J . P . Williams, Israel. J. Chem. 21, 3 (1981) .11 . S . Mann and R . J . P. Williams, J. Chem . Soc. Dalton Trans . 311 (1983) .12 . S . Mann, M . J . Kime, R . G . Ratcliffe, and R . J . P . Williams, J. Chem. Soc. Dalton

Trans. 771 (1983) .13 . B . S . C . Leadbeater, Protoplasma 98, 241 (1979) .14, B . E. Volcani, in Silicon and Siliceous Structures in Biological Systems, B . E .

Volcani and T . L. Simpson, Eds ., Springer Verlag, Berlin, 1981 .15 . P . Westbroek, E . W. de Jong, P . van der Wal, A. H . Borman, J . P. M . de Vrind, D .

Kok, W . C . de Bruyn, and S . B . Parker, Phil. Trans. R . Soc. London B 304, 435(1984) .

16. G . C . Ford, P . M . Harrison, D . W . Rice, J . M. A . Smith, A. Treffry, J . L. White,and J . Yariv, Phil. Trans. P. Soc. London B 304, 551 (1984) .

17 . S . Weiner and W . Traub, FEBS Lett. 111(2), 311 (1980) .

Received May 1986