Inorganic Chemistry in Biology and Medicine - American Chemical

16 High Affinity Iron Transport in Microorganisms Iron(III) Coordination Compounds of the Siderophores Agrobactin and Parabactin

Downloaded by PEPPERDINE UNIV on September 26, 2017 | http://pubs.acs.org Publication Date: December 22, 1980 | doi: 10.1021/bk-1980-0140.ch016

J. B. NEILANDS, T. PETERSON, and S. A. LEONG Department of Biochemistry, University of California, Berkeley, CA 94720

The insolubility of free ferric ion at physiological pH (K = [Fe ][OH ] = 10 39) has required the evolution of special mechanisms in aerobic and facultative anaerobic microbial species for the assimilation of this critically important metal ion. Two pathways have been defined, namely, low affinity and high affinity. The former, still poorly understood, is comparatively inefficient and non-specific. The high affinity system, aspects of which will be the subject of this paper, is itself comprised of two parts: relatively low molecular weight (5001500 daltons), virtually ferric ion specific ligands termed siderophores, and the matching, membrane-associated receptor complex which recognizes and transports the siderophore in its iron laden form. Recent reviews regarding the operation and biomedical significance of the high affinity system, in a l l its variant forms among the bacteria and fungi, are available (1,2,3). Siderophores as chemical entities can generally be classed as either hydroxamates or catechols. Ferrichrome and enterobactin are prototypical members of the two classes, r e s p e c t i v e l y . The 3+

-

3

-

t r i - c a t e c h o l siderophore, e n t e r o b a c t i n , i s o f s p e c i a l i n t e r e s t s i n c e i t has been demonstrated repeatedly that i t can supply i r o n to b a c t e r i a i n the presence o f c e r t a i n f e r r i c tri-hydroxamate type siderophores ( K = l O ^ ) not u t i l i z e d by the organisms (k) . In 1975 T a i t i s o l a t e d a p u t a t i v e siderophore from c u l t u r e s o f Micrococcus (now Paracoccus) d e n i t r i f i c a n s derepressed f o r i r o n and proposed f o r i t s s t r u c t u r e the compound shown i n F i g u r e 1 (R=H), which he c a l l e d "Compound I I I " . Recently (6_) we obtained agrobactin from the phytopathogen Agrobacterium tumefaciens and c h a r a c t e r i z e d i t as an analogue o f Compound I I I c o n t a i n i n g three residues o f 2,3-dihydroxybenzoic a c i d and the o x a z o l i n e form o f the c e n t r a l l y attached r e s i d u e o f L-threonine (Figure 2, R=0H). The protonated oxazoline was shown t o open slowly i n aqueous media t o a f f o r d a g r o b a c t i n A (Figure 1, R=0H). These f i n d i n g s i n s p i r e d a r e i n v e s t i g a t i o n o f "Compound I I I " and l e d t o the d i s covery (j[5_8) o f the oxazoline r i n g i n t h e siderophore from P. d e n i t r i f i c a n s , now renamed p a r a b a c t i n ( F i g u r e 2, R=H). By 0

f

0-8412-05 8 8-4/ 80/47-140-263$05.00/0 © 1980 American Chemical Society Martell; Inorganic Chemistry in Biology and Medicine ACS Symposium Series; American Chemical Society: Washington, DC, 1980.


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AND

H Figure 1.

Parabactin

R = H; agrobactin A, R = OH

Martell; Inorganic Chemistry in Biology and Medicine ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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16.

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E T AL.

Agrobactin

265

and Parabactin


analogy with the agrobactin-agrobactin A p a i r , the open form f o r mula p r e v i o u s l y assigned t o "Compound I I I " has now been designated p a r a b a c t i n A (Figure 1, R=H). The t e r t i a r y Ν atom o f the oxazoline r i n g i n p a r a b a c t i n and agrobactin provides a b i n d i n g s i t e f o r the s i x - c o o r d i n a t e f e r r i c ion. I n s p e c t i o n o f molecular models o f agrobactin suggests two p o s s i b l e modes o f f e r r i c ion- complexation t o the 2,3-dihydroxyphenyloxazoline r i n g system, v i z . , v i a the c a t e c h o l groups o r , a l t e r n a t i v e l y , through the t e r t i a r y Ν and the orthohydroxyphenyl f u n c t i o n . The data r e p o r t e d h e r e i n i n d i c a t e t h a t i n the case o f both siderophores b i n d i n g i s t o the oxazoline n i t r o g e n and t h a t the conformation and c o n f i g u r a t i o n o f the f e r r i c complexes may be as d e p i c t e d i n Figure 3. I.

S t r u c t u r e and P r o p e r t i e s

A. P r e p a r a t i o n o f Siderophores. P u b l i s h e d methods were used f o r the i s o l a t i o n o f a g r o b a c t i n (6) , agrobactin A (6_), p a r a b a c t i n (J}), e n t e r o b a c t i n (^_), r h o d o t o r u l i c a c i d (10), ferrichrome ( l l ) and ferrichrome A (12). P a r a b a c t i n A was obtained by a s y n t h e t i c procedure, t o be reported s e p a r a t e l y . The c a t e c h o l type s i d e r o phores were checked f o r p u r i t y by t h i n l a y e r chromatography on s i l i c a g e l i n k:l chloroform:methanol and by measurement o f the absorption i n t e n s i t y o f the band i n the near u l t r a v i o l e t . Although both p a r a b a c t i n and agrobactin can form hydrochlorides (pKa = 2.3), the siderophores were obtained by e x t r a c t i o n i n t o e t h y l acetate a t n e u t r a l pH and were hence i n the unprotonated form. 1

B.

Ligand Deprotonation

Curves and Proton

Stoichiometry.

a. D i r e c t T i t r a t i o n with F e r r i c C h l o r i d e . I n agrobac t i n the b i n d i n g o f f e r r i c i o n i n the t r i - c a t e c h o l mode would be expected t o r e l e a s e s i x protons. A l t e r n a t i v e l y , c h e l a t i o n t o two catechols p l u s the £-hydroxyphenyloxazoline group would generate only f i v e protons p e r mole. E x a c t l y 2 ymoles o f siderophore were weighed on a Cahn G-2 e l e c t r e b a l a n c e and d i s s o l v e d i n 1-2 ml o f ethanol i n a 10 ml beaker. A f t e r the a d d i t i o n o f 0.1 ml o f meth anol c o n t a i n i n g 2 ymoles o f f e r r i c c h l o r i d e the s o l u t i o n was d i l u t e d with water t o ca. 5 ml and standard 0.100 Ν NaOH introduced with the r e c o r d i n g t i t r a t i o n apparatus (13). The complexes, which were i n i t i a l l y b l u e , became purple and then wine i n c o l o r as the t i t r a t i o n progressed i n t o n e u t r a l pH. From the data shown i n Figure k i t i s apparent t h a t between h and 5 protons p e r mole were r e l e a s e d a t n e u t r a l pH. A l s o s t r i k i n g was the f a c t t h a t the deprotonation curves f o r agrobactin and p a r a b a c t i n remained superimposable a t a l l except very a l k a l i n e values o f pH. 9


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266

Figure 4. Titration of agrobactin and parabactin in the presence of an equimolar quantity of ferric chloride. The siderophores differed significantly only at very high pH, where ( ) agrobactin displayed a new buffer zone not present in ( ) parabactin.


16.

NEILANDS

Agrobactin

E T AL.

and

267

Parabactin

b. Ligand Exchange. The well-known tendency o f f e r r i e catecholates t o undergo i n t e r n a l o x i d a t i o n - r e d u c t i o n a t lower values o f pH (lJi>l£)> concern f o r the s t a b i l i t y o f the oxazoline moieties , and the opportunity t o diminish the proton discrepancy between t r i - c a t e c h o l ys_ b i - c a t e c h o l - p l u s - o x a z o l i n e b i n d i n g i n agrobactin from 1 i n 6 down t o 1 i n 3 prompted us t o supply i r o n to the siderophores v i a a l i g a n d exchange r e a c t i o n o f the type: Agrobactin + f e r r i c rhodotorulate = f e r r i c agrobactin +


2 or 3 H

+

+ rhodotorulic acid.

Rhodotorulic a c i d , a hydroxamate type siderophore, binds 2 / 3 mole f e r r i c i o n t o give an uncharged, orange c o l o r e d c h e l a t e which i s f u l l y formed at n e u t r a l pH ( 1 0 ) . T i t r a t i o n s were performed as above except t h a t 2 ymoles o f f e r r i c c h l o r i d e and 3 ymoles o f r h o d o t o r u l i c a c i d were mixed and n e u t r a l i z e d p r i o r t o i n t r o d u c t i o n o f 2 ymoles o f c a t e c h o l type siderophore d i s s o l v e d i n ethanol. The equivalents o f standard a l k a l i r e q u i r e d t o n e u t r a l i z e the s o l u t i o n were then noted. Upon i n t r o d u c t i o n o f 2 ymoles o f agrobactin the c o l o r o f the n e u t r a l f e r r i c hydroxamate s o l u t i o n changed from orange t o blue and the pH f e l l t o 3 . 6 . E x a c t l y 3 . 9 8 ymoles o f a l k a l i were r e q u i r e d t o r a i s e the pH t o the end p o i n t o f pH 6 . 5 t o 7 , c o r r e s ponding t o 2 moles H per F e 3 . I n the case o f p a r a b a c t i n , the pH went t o j u s t l e s s than k.O upon a d d i t i o n o f the c a t e c h o l and the c o l o r o f the s o l u t i o n was brown, thus i n d i c a t i n g the presence o f some f e r r i c hydroxamate. R e s t o r a t i o n o f the pH t o 6 . 5 t o 7 which again appeared t o be an end p o i n t , r e q u i r e d 1 . 7 H p e r F e 3 . F o r agrobactin A, the pH f e l l t o k.k and the s o l u t i o n , although wine c o l o r e d , upon back t i t r a t i o n was s t i l l b u f f e r i n g s t r o n g l y i n the pH range 7 - 8 . From these data we conclude that agrobactin and p a r a b a c t i n form s i m i l a r c o o r d i n a t i o n compounds with f e r r i c i o n i n which one o f the atoms l i n k e d t o the i r o n i n the c e n t r a l b i d e n t a t e p o r t i o n o f the complex i s the t e r t i a r y Ν o f the oxazoline r i n g . At n e u t r a l pH agrobactin A, i n c o n t r a s t t o the t r i - c a t e c h o l e n t e r o b a c t i n ( l 6 ) , appeared t o be not f u l l y coordinated t o the f e r r i c i o n . S i m i l a r experiments were performed by l i g a n d exchange with the 1 : 1 f e r r i c complex o f n i t r i l o t r i a c e t a t e . The t o t a l proton y i e l d s p e r i r o n t o the n e u t r a l pH zone were ^ . 7 and k.6 f o r agro b a c t i n and p a r a b a c t i n , r e s p e c t i v e l y . Thus i n t r o d u c t i o n o f the i r o n ( I I I ) , e i t h e r d i r e c t l y o r by l i g a n d exchange, gave complexes which f o r both agrobactin and p a r a b a c t i n should r e s u l t i n d i v a l e n t anions at n e u t r a l pH. +

+

5

+

+

C. Net E l e c t r i c a l Charge. The i r o n complexes o f a g r o b a c t i n , agrobactin A, p a r a b a c t i n and e n t e r o b a c t i n were prepared by neu t r a l i z a t i o n o f the ligands i n the presence o f f e r r i c c h l o r i d e and t h e i r e l e c t r o p h o r e t i c m o b i l i t i e s compared with t h a t o f ferrichrome


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A on paper at pH 6 . 6 i n 0 . 1 μ phosphate b u f f e r . recorded i n Table I.

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The r e s u l t s

are

Table I E l e c t r o p h o r e t i c M o b i l i t y of Siderophore m

Complex

Iron ( i l l ) Complexes

Net Negative Charge

Relative Mobility


pH

6.6

Fe ( I I I ) a g r o b a c t i n

687

2

0.85

Fe ( I I I ) agrobactin A

10k

2-3

1.03

Fe ( I I I ) p a r a b a c t i n

671

2

0.87

Fe ( I I I ) e n t e r o b a c t i n

719

3

1.20

1052

3

1.00

Ferrichrome

A

While the m o b i l i t y of substances on paper i n an e l e c t r i c f i e l d i s known t o be i n f l u e n c e d by a number of f a c t o r s , charge and mass are c e r t a i n l y the two most s i g n i f i c a n t parameters a f f e c t i n g the r a t e o f m i g r a t i o n . The data i n Table I i l l u s t r a t e t h a t f e r r i c agrobactin and f e r r i c p a r a b a c t i n are mononuclear and move at comparable r a t e s ; the s l i g h t l y enhanced r a t e of f e r r i c parabac t i n would be expected i n view of i t s lower molecular weight, pro v i d e d both complexes bore 2 negative charges. I f f e r r i c entero b a c t i n were reduced from a net charge o f 3 " t o 2 " , i t s a n t i c i p a t e d m o b i l i t y r e l a t i v e t o ferrichrome A would be - 0 . 8 . The somewhat g r e a t e r r a t e s shown f o r f e r r i c agrobactin and f e r r i c p a r a b a c t i n , 0 . 8 5 and 0 . 8 7 , r e s p e c t i v e l y , are i n i n v e r s e p r o p o r t i o n t o t h e i r lower molecular weights. F e r r i c agrobactin A could be speculated to c a r r y between 2 and 3 charges/mole at t h i s pH. A l l of the f e r r i c catechols l i s t e d i n Table I were wine c o l o r e d at pH 6 . 5 · In c o n t r a s t , an i r o n complex of p a r a b a c t i n A was purple at both pH 6 . 5 and 7.U, and i t d i s p l a y e d an unusually low m o b i l i t y , thus i n d i c a t i n g i t t o be incompletely formed and/or m u l t i - n u c l e a r . In sum, the e l e c t r o p h o r e s i s experiments support the concept of i r o n ( i l l ) b i n d i n g to the oxazoline Ν i n both agrobactin and p a r a b a c t i n t o g i v e , i n each case, mononuclear d i - a n i o n i c com plexes . D. E l e c t r o n i c Absorption Spectra. The absorption s p e c t r a of the complexes formed by exchange from f e r r i c n i t r i l o t r i a c e t a t e were determined i n 0 . 1 M phosphate, pH 7.h with a Beckman Model 25 r e c o r d i n g spectrophotometer over the range i i 0 0 - 7 0 0 nm. Agro b a c t i n and p a r a b a c t i n gave wine c o l o r e d i r o n complexes with a broad adsorption band centered at about 5 0 0 nm. At pH l.h the 9

9


16.

Agrobactin

NEILANDS E T A L .

269

Parabactin

approximate aga* f o r a g r o b a c t i n was U . l at 5 0 5 nm; f o r p a r a b a c t i n the t e n t a t i v e f i g u r e was 3 . 3 at 5 1 2 nm. Tait r e p o r t e d a value at pH 7.U o f 3 . 5 at 5 1 5 nm f o r h i s "Compound I I I " , which we bel i e v e t o have the s t r u c t u r e shown i n F i g u r e 2 , R=H. The absorpt i o n maximum f o r f e r r i c p a r a b a c t i n A was s h i f t e d s u b s t a n t i a l l y t o the red and l a y at 5 3 5 nm. Since the s p e c t r a o f f e r r i c a g r o b a c t i n and f e r r i c p a r a b a c t i n do show minor d i f f e r e n c e s , a number o f experiments were t r i e d i n which an attempt was made t o mimic these d i f f e r e n c e s v i a examinat i o n o f the 1 : 3 f e r r i c complexes o f model s a l i c y l and 2 , 3 - d i h y droxyphenyloxazolines and the 1 : 1 : 1 f e r r i c complexes o f the model oxazolines with T a i t s "Compound I I " , N ,N°-bis-(2,3-dihydroxybenzamido)spermidine. F e r r i c t r i s - s a l i c y l o x a z o l i n e p r e c i p i t a t e d upon i t s formation by the a d d i t i o n o f three equivalents o f a l k a l i . The n e u t r a l complex was d i s s o l v e d i n ethanol t o g i v e an orange c o l o r e d s o l u t i o n with a ^ = U.5 at the maximum, U 6 5 nm. A specimen o f f e r r i c t r i s 2,3-dihydroxyphenyloxazoline had a maximum at 5 2 0 nm with a ^ o f 2 . 8 at pH 7 · 5 ; at pH 1 0 , where the complex was f u l l y formed, the a^iM was k.6 and the maximum had s h i f t e d t o U 9 5 nm. These data, and those obtained by admixture o f the oxazolines w i t h T a i t s Compound I I , were not p a r t i c u l a r l y i l l u m i n a t i n g as regards the mode o f i r o n complexation i n the n a t u r a l products. Examination o f the complexes i n the u l t r a v i o l e t might have proved i n s t r u c t i v e , but t h i s aspect was not pursued. f


and

1

1

E. C i r c u l a r Dichroism. The siderophores s t u d i e d h e r e i n cont a i n one b i d e n t a t e l i g a n d mounted on an o p t i c a l l y a c t i v e subs t i t u e n t , namely, L-threonine or i t s o x a z o l i n e . Thus the p a r t i c u l a r type o f c o o r d i n a t i o n isomer considered by Corey and B a i l a r ( 1 7 ) c o u l d r e s u l t i n a chelate chromophore which i s capable o f r o t a t i o n o f plane p o l a r i z e d l i g h t . The c i r c u l a r dichroism s p e c t r a o f the f e r r i c d e r i v a t i v e s o f the two siderophores, t h e i r "A" analogues, and e n t e r o b a c t i n were determined i n a s u i t a b l y equipped Cary Model 6 0 s p e c t r o p o l a r i meter. The data are recorded i n F i g u r e 5 · I t i s apparent t h a t the curve f o r f e r r i c a g r o b a c t i n A resembles t h a t o f e n t e r o b a c t i n , which i s known t o y i e l d a A , c i s complex w i t h f e r r i c i o n ( l 8 ) . I n c o n t r a s t , the curves f o r f e r r i c agrobactin and f e r r i c p a r a b a c t i n suggest c l e a r l y t h a t i n t h i s case the c o n f i g u r a t i o n around the i r o n i s predominantly a l e f t - h a n d e d p r o p e l l e r , i . e . , A,cis_, as i n ferrichrome (l£). S t e r i c c o n s t r a i n t s r u l e out the p o s s i b l e p r e s ence o f geometrical isomers o f the t r a n s v a r i e t y . F.

Formation

Constant.

a. E q u i l i b r a t i o n w i t h Ferrichrome. The data i n F i g u r e 6 i l l u s t r a t e the a b i l i t y o f a g r o b a c t i n to remove i r o n from f e r r i chrome under the s p e c i f i c experimental c o n d i t i o n s employed. S i m i l a r r e s u l t s were obtained w i t h p a r a b a c t i n , e n t e r o b a c t i n and a syn-


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τ

1

300

350

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1

400

AND

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Γ

450

500

nm Figure 5. Circular dichroism spectra of approximately 0.2mM solutions of A, ferric enterobactin; B, ferric agrobactin; C, blank; D, ferric agrobactin A; and E, ferric parabactin in 0.1 M phosphate pH 7.4

0.4|

1

1

1

1

ι 10

ι 20

ι 30

ι 40

Γ

o.ih

I Ο

Ι 50

Hours

Figure 6.

Equilibration of 0.1 mM ferrichrome and 0.1 mM agrobactin in 50% ethanol-50% WmU NaHEPES buffer pH 7.2 at 25°C


16.

NEILANDS E T A L .

Agrobactin

and

271

Parabactin

t h e t i c analog o f e n t e r o b a c t i n , c i s . 1 , 5 , 9 - t r i s ( 2 , 3 - d i h y d r o x y b e n z a mido)cyclododecane (20). I t i s apparent t h a t a l l o f these c a t e c h o l compounds are thermodynamically capable o f c a p t u r i n g i r o n from f e r r i c h r o m e , which i s r e p o r t e d t o have K = 1 0 2 9 · 1 ( 2 l ) . Since i n each case the i r o n was completely t r a n s f e r r e d , we e s t i mate t h a t the Kf f o r the t r i - c a t e c h o l s must be at l e a s t s e v e r a l orders o f magnitude greater than those r e p o r t e d f o r the t r i hydroxamate type siderophore l i g a n d s .


f

b. E q u i l i b r a t i o n w i t h F e r r i c E n t e r o b a c t i n . Having e s t a b l i s h e d t h a t the c a t e c h o l type siderophores based on spermi dine are s u p e r i o r i r o n ( i l l ) complexing agents ( i n comparison with ferrichrome) we next sought t o c o n t r a s t them w i t h enterobac tin. The r e l a t i v e a v i d i t y o f a g r o b a c t i n , p a r a b a c t i n and entero b a c t i n f o r i r o n ( i l l ) was estimated by observation o f the progress of the f o l l o w i n g r e a c t i o n s from l e f t t o r i g h t : F e r r i c enterobactin + agrobactin + H

+

= ferric

agrobactin

+ enterobactin F e r r i c enterobactin + parabactin + H

+

= ferric

parabactin

+ enterobactin F e r r i c agrobactin + e n t e r o b a c t i n = f e r r i c e n t e r o b a c t i n + agrobactin +

H

+

F e r r i c parabactin + enterobactin = f e r r i c enterobactin +

+ parabactin + Η A t i t r a t i o n v i a l was charged w i t h 0 . 5 ml e t h a n o l , 2 ymoles o f c a t e c h o l type siderophore i n 0.2 ml e t h a n o l , 2 ymoles o f F e C l ~ i n 0.1 ml methanol and 0 . 5 ml o f water. The pH was brought t o aDout 7 with standard 0.1 Ν NaOH and 0.2 ml c o n t a i n i n g 2 ymoles o f com p e t i n g c a t e c h o l type siderophore was then added. I f necessary, the pH was r e a d j u s t e d t o ~ 7 · Upon adding e i t h e r a g r o b a c t i n or p a r a b a c t i n t o n e u t r a l s o l u t i o n s o f f e r r i c e n t e r o b a c t i n there was l i t t l e change i n pH. How ever, mixing o f e n t e r o b a c t i n w i t h e i t h e r f e r r i c a g r o b a c t i n or f e r r i c p a r a b a c t i n caused the pH t o f a l l t o l e s s than 6 and ca. 1 t o 2 ymoles o f a l k a l i were r e q u i r e d to n e u t r a l i z e the s o l u t i o n s . The n e u t r a l s o l u t i o n s were allowed t o stand at room tempera t u r e f o r 1 hr and then s u b j e c t e d t o paper e l e c t r o p h o r e s i s f o r 30 minutes at pH 6.6. A l l f o u r mixtures separated i n t o about equal amounts o f the two f e r r i c complexes ; a s i m i l a r p a t t e r n was observ ed a f t e r i n c u b a t i o n f o r 2h hours at room temperature. We conclude from these r e s u l t s t h a t l i g a n d exchange was r a p i d and t h a t a l l


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three siderophores have about equal a f f i n i t y f o r i r o n . B u f f e r i n g by the _o-hydroxyl aromatic f u n c t i o n s , which may have p K values

Inorganic Chemistry in Biology and Medicine - American Chemical

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