The Role of Inorganic Chemistry in Cellular Mechanisms of Host

23 The Role of Inorganic Chemistry in Cellular Mechanisms of Host

Downloaded by FUDAN UNIV on February 25, 2017 | http://pubs.acs.org Publication Date: May 5, 1997 | doi: 10.1021/ba-1997-0253.ch023

Resistance to Disease James K. Hurst Department of Chemistry, Washington State University, Pullman, WA 99164-4630

Phagocyticcellsassociated with host resistance to disease appear to be capable of generating a variety of inorganic oxidants that function as microbicidal agents. In neutrophils and related cells containing myeloperoxidase, hypochlorous acid is a primary microbicide; the nature of oxidants produced by other types of phagocytic cells are less well characterized, but may involve metal-mediated reactions of H O or intermediary formation of peroxonitrite (ONO ) ion. The biochemical role of these oxidants is reviewed from the perspective of mechanistic inorganic chemistry. Results from recent studies suggesting unique roles for HCO and CO in these processes are also described. 2

2

-

2

-

3

2

XJPON E N C O U N T E R I N G A B A C T E R I U M , phagocytic white blood cells undergo a progression of biochemical transformations that lead to isolation of the organ ism within a highly inimical environment (I). Prominent among these transfor mations is activation of a respiratory chain that catalyzes the one-electron reduction of oxygen to superoxide ion (2). As discussed herein, the subsequent fate of ion is the subject of considerable debate and experimental investi gation. Remarkably, the conceptual basis of almost all current discussions of the identities of the ultimate toxins and their modes of action is the seminal mechanistic work (3-7) on the redox chemistry of main group elements carried out by Henry Taube and associates four decades ago. Not only do the general mechanistic principles laid out in these studies serve as a design model for investigating similar reactions in the more complex biological arena, but his early research on the chemistry of oxygen, hydrogen peroxide, and the oxides of chlorine and nitrogen, i n particular, remains directly relevant to current © 1997 American Chemical Society

Isied; Electron Transfer Reactions Advances in Chemistry; American Chemical Society: Washington, DC, 1997.

399

400

E L E C T R O N TRANSFER REACTIONS

mechanistic issues concerning cellular disinfection processes. Notable among the latter are his studies on the reaction between HOC1 and H 0 to form elec tronically excited { Δ)0 (4-5) and reactions between H N 0 and H 0 to form peroxonitrous acid ( O N 0 H ) and its subsequent isomerization to nitric acid (6). 2

λ

2

2

2

2

2

2


Phagocytosis: A Closer Look The events comprising bacterial phagocytosis are illustrated stylistically i n Fig ure 1. Binding at receptor sites on the cellular membranes of phagocytic cells elicits a secondary-messenger cascade that leads ultimately to phosphorylation of specific cytosolic proteins (8). This phosphorylation triggers assembly of cytosolic and membrane-localized proteins to form a functioning electron transport chain. The respiratory chain appears to be unusually simple, contain ing only F A D and b-type hemes as redox components {8-9). It is vectorially organized across the plasma membrane, so that electrons from cytosolic donors ( N A D P H ) are translocated to the external environment where the 0 reduc tase site is located (10). Simultaneously with respiratory activation, the plasma membrane invaginates, surrounding the bacterium and eventually pinching off 2

OPSONIZED MICROBE

PHAGOSOME

NEUTROPHIL

Figure 1. Diagram of phagocytosis by neutrophih. [1] Binding of the opsonized bacterium at receptor sites initiates a secondary-messenger cascade that evokes the respiratory burst; [2] phagocytosis proceeds by membrane invagination, which ultimately pinches off [3] to form the new vacuole (phagosome). Simultaneous degranulation leads to both extracellular secretion and intraphagosomal accumu lation of granule components. MPO means myeloperoxidase. (Reproduced with permission from reference 10. Copyright 1989 CRC Press.)


23.

HURST

Mechanisms of Host Resistance to Disease

401

to form an internalized vacuole (phagosome) containing the entrapped bac terium. Because the membrane everts in this process, the respiratory chain is now oriented to generate Ό within the phagosome, the bulk of which appears to undergo nonenzymatic disproportionation to form H 0 . In neutrophils (the predominant type of phagocytic white blood cells), these events are accompa nied by simultaneous migration of lysosomal granules containing enzymes and related biopolymers from the cytosol to the phagosomal membrane, where the membranes fuse and the lysosomal contents are discharged into the phagoso mal volume (J). Among the lysosomal enzymes is a unique peroxidase (myeloperoxidase, M P O ) that is capable of catalyzing the two-electron oxida tion of CI" to HOC1 (II). This enzyme is present in astonishingly high concen trations, comprising 2-5% of the total weight of the neutrophil (12). The entire process from binding to lysosomal degranulation and killing of the bacterium requires only a few minutes; in many cases the bacterial cell morphology is indistinguishable from normal viable cells, indicating that gross physical dis ruption of the bacterial envelope is not the cause of death (13-14). Although the immediate source of electrons for 0 reduction is N A D P H , this reductant is cyclically regenerated by glucose oxidation via the hexose monophosphate pathway (I). Consequently, the oxidized respiratory end prod uct is C 0 , which can be expected to rise severalfold above the normal physio logical levels of —25 m M during the course of stimulated respiration. The 0 reductase site is thought to be the heme prosthetic group of the cytochrome (8, 9). Although structural characterization is incomplete (e.g., evidence suggesting that the cytochrome contains two hemes per F A D is accumulating), resonance Raman spectral analyses indicate that the environment of the heme (or both hemes) is 6-coordinate low spin in both Fe and F e oxidation states (14,15). This strong axial ligation precludes direct 0 binding to the heme iron, forcing the reaction to proceed by an "outer-sphere" mechanism (16), hence, i n oneelectron steps. Thus, formation of Ό as the immediate product is ensured. Two lines of evidence indicate that oxidative mechanisms are important to bacterial killing. One is that many bacteria are killed much less efficiendy by neutrophils under anaerobic conditions than in the presence of oxygen (17,18). The other is that individuals with a congenital defect known as chronic granu lomatous disease, characterized by a defective •Oj-generating oxidase but oth erwise normal phagocytic capabilities, suffer chronic life-threatening bacterial infections (I). Thus, formation of 0 -derived oxidants is essential to cellular defense mechanisms. In contrast, the role of M P O has been controversial. Individuals with hereditary M P O deficiency, characterized by the absence of a functional peroxidase but an otherwise normal phagocytic response, exhibit only minor clinical manifestations of this disease (19, 20). Nonetheless, M P O deficient neutrophils are by several criteria considerably less effective than normal neutrophils in in vitro studies of bactericidal potency (19). In addition to its unique capacity to efficiently catalyze C l " oxidation, M P O catalyzes two-electron oxidation of other halides and pseudohalides (21, 2


2

2

2

2

2

11

m

2

2

2



402


22), as well as one-electron oxidation of organic substrates (23). This diversity of catalytic capability calls into question its true physiological function. How ever, based upon competition studies, the preferred substrate in normal physi ological environments appears to be CI" (22). Furthermore, evidence consistent with direct chlorination within the phagosome has been obtained using a recoverable fluorescent probe (Q. Jiang, unpublished observations). In the probe studies (Figure 2), fluorescein was attached to 0.5-2.0-μπι carboxyderivatized polyacrylamide spheres via cystamine linker groups. Following opsonization, that is, targeting with serum-derived antibodies and comple ment, the particles were avidly phagocytosed by isolated neutrophils. Fluores cence changes consistent with chlorination of the probe were observed during and immediately following phagocytosis. The dye was subsequently recovered in near-quantitative yield by cell lysis, followed by its release from the particle by cleavage of the cystamine disulfide bond upon addition of a thiol. The only reaction products detected by H P L C analysis co-chromatographed with and had spectroscopic features analogous to authentic samples of mono- and dichlorofluorescein thiols; these structures have now been confirmed by ion electrospray mass spectrometry (D. Ε Barofsky and D. A. Griffin, unpublished observations). Thus, MPO-catalyzed intraphagosomal chlorination undoubt edly occurs. Since HOC1 is freely diffusible from the enzyme active site (JI) and the same ring-chlorinated products are obtained from reaction between H O C 1 and fluorescein (24), one infers that H O C l can be formed within the phagosome. To summarize, the most probable primary set of reactions leading to for mation of bactericidal agents in normal neutrophils is enzyme-catalyzed oneelectron reduction of 0 by glucose, followed by disproportionation of the res piratory end-product Ό to H 0 and its MPO-catalyzed oxidation of C l " to H O C l (Figure 3). Alternatively, H O C l could be formed by direct reaction of Ό with M P O , yielding compound III (the F e - O adduct) as an intermedi ary species (25, 26) 2

2

2

2

m

2

MPO(Fe ) + Ό m

2

2

+ H - > compound III ( F e - O H ) +

m

followed by its one-electron reduction by a second 0 ferryl π-cation): compound III + Ό

2

2

2

to give compound I (a

+ H -> compound I ( F e ^ O π-cation) + H 0 + 0 +

2

2

which is the active form of the catalyst (27) compound I + C l " + H - » M P O + H O C l +

and the same product formed by direct reaction of M P O with H 0 . A n addi tional function of Ό may be to prevent accumulation of compound II, a ferryl 2

2

2



23.

HURST


403

separation & analysis

Figure 2. Recoverable probes for detecting chlorination and other postphagocyMc events; the symbolflrefers to thefluoresceinmoiety. In a typical experiment, the fluorescein-labeled particles are vortex-mixed with neutrophils to elicit binding, and the subsequent changes influorescenceproperties of the dye are monitored. The dye is then recovered for chemical analysis by (1) homogenizatkm of the neutrophils to release the particles, (2) cleavage of the linker disulfide bond with dithiothreitol to release the dye, and (3) centiifugation to remove cell debris and the now-unlabeled polyacryhmide beads. (Reproduced with permission from reference 78. Copyright 1993 Plenum Press.)


404

1


glu

co

NADP

o-

+

2

NADPH-

2

0

2

H 0 2

cr

m

2

HOCl

Figure 3. Flow diagram for respiratory generation of HOCl and C0 from glucose and 0% Catalysis by [1] the enzymes involved in the hexose monophosphate shunt, [2] the NADPH oxidase, and [3] myeloperoxidase. The symbol glu refers to glu cose. Downloaded by FUDAN UNIV on February 25, 2017 | http://pubs.acs.org Publication Date: May 5, 1997 | doi: 10.1021/ba-1997-0253.ch023

2

form of the catalyst that is incapable of oxidizing C l " , by reducing it to the native ferric M P O state (28) and thereby returning it to the catalytic cycle, viz.: compound II (Fe =0) + Ό IV

2

+ 2 H -» MPO + 0 +

2

+ H 0 2

In any event, directly or indirecdy, Ό is an appropriate substrate for M P O catalyzed formation of H O C l . Hypochlorous acid might itself be the bacterici dal agent produced by these cells or, alternatively, might react with endoge nous amines to form the corresponding N-chloramines, which are also potent bactericides. This issue of the identity of the ultimate toxin is presently unre solved (10, 29) but will not affect our discussion of bactericidal mechanisms because the chemical principles governing reactivities are the same for the two oxidants. 2

MPO-Dependent Mechanisms: The Chemical Basis for HOCl Toxicity Hypochlorous acid is highly toxic to prokaryotic cells (1,19). A comparison of in vitro bactericidal assays for a prototypic bacterium, Escherichia colt, using vari ous oxidants under roughly comparable conditions is given i n Table I. In this comparison, H O C l is at least lO^fold more toxic than the more strongly oxidiz ing H 0 , O N 0 ion, and * O H radical (30). Approximately 10 molecules of H O C l are required to kill one E. coli cell (31); this means that 1 m L of a prop erly distributed commercial bleach solution (5% HOCl) would be sufficient to kill 5 g of cells! Other oxidants, for example, H 0 in the presence of C u and a reducing agent (32), and H C O 3 radical (33), also exhibit high toxicity, approaching that of H O C l (Table I). How can we understand these wide varia tions i n toxicity? Since the oxidant quantities required to inflict lethal damage in the more effective bactericidal systems are remarkably small, cellular death must be associated with destruction of a limited number of vulnerable sites within the bacterium. Correspondingly, toxicity is associated with oxidant 2

2

8

2

2

2


2 +

23. HURST Mechanisms of Host Resistance to Disease Table I. LD^ for Inorganic Toxins Against Escherichia colt in Suspension Cell Density (viable celh/mL)

Toxin

U> (molecules/ cell)

HOCl

0.4-5.0 x l O ^

1-12

5xl0

>3 χ 10

73

9 6

3

6

6

2

6

7

3

2

9

2

2+

2

N O T E : Values are for strain A T C C 25922 in 0.05-0.10 M phosphate, p H 7.4, containing 0.15 M NaCl, unless otherwise specified. LDQQ is dose level required to kill 90% of the cells. Relative toxi city values are scaled to H O C l at pH 7.4 ( L D ^ = 5 χ 10 HOC1/E. coli). E ° values are standard 8

7

reduction potentials (vs. NHE) at pH 7.0. NaCl absent.

û

*pH dependent (pH 5.0-7.4). [Cu ] and [ascorbate] dependent.

c

2+

0.l M carbonate buffer, pH 6.5-7.4.

d

^Anaerobic media. /Calculated from E°('C0 7C03 -) = 1.6 V (75) and the proton dissociation constants (76). 2

3

^Calculated from £°(Cu /Cu ) = 0.16 V (73) and the cuprous chloride association constant (77). 2+

+

selectivity for these target sites, rather than its capacity to inflict massive, non specific oxidative damage to the cells. The key to understanding the toxicity of an oxidant therefore lies in understanding its inherent chemical reactivity. E. coli are killed within 100 ms after exposure to lethal doses of H O C l (31). For this oxidant, the vulnerable sites are clearly among the more reactive biomolecules. A useful model system to explore the reactivity of H O C l is its oxida tion of H 0 : 2

2

HOCl + H 0 2

2

-» 0 +H 0 +H + Cl" 2

2

(1)

+

A crucial early observation by Cahill and Taube (4) was that both Ο atoms in 0 were obtained from H 0 , which precludes any reasonable radical reaction mechanism. One-electron oxidations by H O C l are also unlikely on thermody namic grounds since they require formation of high-energy Ό Η or C l radicals as reaction products (34). As illustrated by Taube's early mechanistic studies (3-7) on atom transfer reactions, two-electron oxidation requires some form of incipient bond formation. Rate measurements provided indirect evidence for 2

2

2

e


406


this type of association. Specifically, reaction 1 was dominated by a pathway whose rate law is - d t H O C i y d * = fe[H 0 ] [HOCl] where the subscript ο refers to total reactant concentration. The bimolecular rate constant (k) exhib ited a bell-shaped pH-rate profile indicating that the true reactant pairs are either H O C l and H 0 or OC1" and H 0 (35). The reactions of H 0 with a series of chlorine(+l) compounds for which the dissociable proton was replaced by a nondissociable electron-withdrawing group (X) gave rate laws of the form d[0 ]/df = fc[H0 ][X-Cl], with the rate constant for reaction of tertbutyl hypochlorite approaching the value calculated for reaction between H O C l and H 0 (36). These observations implicate H O C l and H 0 as the true reactants i n the H 0 C 1 - H 0 reaction. What is remarkable about this reaction is that the other possible pairs, namely, H 0 and H O C l , H 0 and O C l , or H 0 and OC1" are unreactive, despite there existing a relatively large thermodynamic driving force for reaction between them (35). [A slow reaction occurs between H O C l and H 0 , the rate law for which is d[0 ]/d* = fc[HOCl][H ][Cl1, independent of the H 0 concentration; the rate is enhanced i n acetate and phthalate buffers, possibly by general acid catalysis (35). In physiological environments, H C O 3 might cause similar effects (D. T. Sawyer, personal communication), although this possibility has not been examined.] The unique feature of the H O C l and H 0 reactant pair is that it combines a relatively electrophilic chlorine atom with a strongly nucleophilic H 0 ion, which favors electrophile-nucleophile interactions of the type shown i n Figure 4. In this model, incipient bond formation is thought to lead to two-electron transfer from the electronegative hydroperoxide oxygen atom to chlorine, lead ing to net oxidation-reduction. Additional supporting evidence consistent with this transition state structure are the observations that (1) di-ierf-butyl hydroperoxide, which has no dissociable proton, is unreactive toward H O C l (D. T. Sawyer, personal communication) and (2) the rate constant for reaction of H 0 decreases proportionately with decreasing electron-withdrawing charac ter of the chlorine substituent group (X), that is, with decreasing electrophilic character of the chlorine atom (Figure 4). Whether a discrete C l O O H interme diate is formed, as has been proposed for the reaction between C l and H 0 in acidic media (4 37), cannot be established from the kinetic data. Based upon these kinetic properties, we would expect H O C l to react pref erentially with nucleophilic centers in biomolecules, and, indeed, this is what is observed. Hypochlorous acid displays an exceptionally wide range of reac tivity toward prototypic biological partners, rapidly oxidizing electron-rich πdelocalized centers such as nitrogen heterocycles (hemes or nucleotide bases), iron-sulfur clusters, and conjugated polyenes (e.g., carotenes), as well as amino acids containing highly polarizable sulfur atoms and amines, while being virtu ally unreactive toward compounds not possessing nucleophilic sites (38). This same selectivity has been shown to extend to biomolecules within bacteria. Specifically, oxidation of sulfhydryl substituents and N-chlorination of 2

2

2

2

2 0

0

2

2

2

2

2

2


2

2

2

2

2

2

2

2

2

+

2

2

-

2

2

2

2

2

9


2

2

23.

HURST

407


k(M-ls-l)

χ

H 0-0

HO-

4.4x107

(CH ) CO-

1.5x106

3

3

£1


9.1x104

The Role of Inorganic Chemistry in Cellular Mechanisms of Host

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