(Received for publication, November 29, 1995)
From the
The terebellid polychaete Amphitrite ornata produces no
detectable volatile halogenated secondary metabolites, but frequently
inhabits coastal marine sediments heavily contaminated with
anthropogenic or biogenic haloaromatic compounds. This animal contains
high levels of two very unusual enzymes, dehalogenating peroxidases. We
have purified and partially characterized one of these
dehaloperoxidases, DHP I. DHP I is a heme enzyme (M = 30,790) composed of two identical subunits (M
= 15,529) and is very rich in the amino
acids aspartic acid (+ asparagine) and glutamic acid (+
glutamine). The enzyme converts trihalogenated phenols, such as
2,4,6-tribromophenol, into dihalogenated quinones. The optimum pH for
this reaction is 5.0. DHP I is also active against di- and
monohalogenated phenols and will oxidize bromo-, chloro-, and
fluorophenols. We have identified similar dehaloperoxidase activities
in other infaunal polychaetes, including halometabolite-producing
species.
Contamination of coastal marine sediments by anthropogenic haloaromatic compounds found in agricultural, industrial, and urban runoff is well known(1, 2) . Less recognized, but common and very widespread sources of haloaromatics are biotic(3, 4, 5) . Among these are species of sediment-dwelling marine polychaetes and hemichordates which produce high levels of volatile brominated secondary metabolites, such as bromophenols, bromopyrroles, bromoindoles, bromohydroquinones, and bromobenzylalcohols(5, 6, 7, 8) , through the action of haloperoxidases(9, 10) . Such worms occupy immense areas of coastal sediments(11, 12, 13, 14, 15) , contaminating them with volatile, malodorous, and toxic bromometabolites. Bromophenols and related compounds are respiratory inhibitors and presumably express their toxicity through inhibition of mitochondrial function. The toxicity of these compounds to vertebrates is well established(16, 17, 18, 19) , but has not been as thoroughly examined in invertebrates. It is clear from field and laboratory studies that sediment contamination with bromoaromatics inhibits recruitment of non-bromometabolite-producing invertebrate species (15) and appears to select for specific organisms which may be resistant to the toxic effects of haloaromatic compounds. Both these resistant species and the bromometabolite producers themselves, which face bromometabolite autotoxicity, must have some means of detoxifying these compounds. Dehalogenating enzymes provide one such detoxification mechanism.
Dehalogenating enzymes
are uncommon in higher organisms. The cytochrome P-450 enzymes are
exceptions, being broadly distributed among animals, plants, fungi, and
bacteria and capable of reductive dehalogenation of alkyl halocarbons
to the corresponding alkanes under anaerobic conditions(20) .
Other oxygenases may also participate in similar reactions, but
haloaromatic compounds present a unique problem due to their relative
stability and toxicity. The terebellid polychaete Amphitrite ornata produces no detectable volatile halometabolites of its own ()and lacks detectable halogenase activity. However, this
animal is often found in beds also inhabited by Notomastus lobatus (Polychaeta) and Saccoglossus kowalewskyi (Hemichordata),
which produce and contaminate sediments with bromophenols and
bromopyrroles(8, 9, 13, 21) . We
examined A. ornata to determine the basis for its tolerance of
haloaromatic compounds and found that, instead of a typical oxygenase,
it produces high levels of two dehalogenating peroxidases. We have
purified and partially characterized dehaloperoxidase I (DHP I), (
)which has a number of unusual properties.
Figure 1: Protein profiles of the Amphitrite ornata dehaloperoxidase I and molecular weight standards fractionated on a sodium dodecyl sulfate-polyacrylamide gel (15% gel). Molecular weights are indicated in thousands.
Dehaloperoxidases are abundant in A. ornata, accounting for roughly 3% of soluble protein recovered in crude extracts (Table 1). Initial chromatography steps provided a 10.1-fold purification of dehaloperoxidase I with loss of 38% of total DHP activity. This major activity loss is accounted for by separation of the two dehaloperoxidases present in crude extract. The two red-colored DHP peaks were always visible in the ion exchange chromatography fractions. Peak 1 eluted at about 25 ml (approximately 0.1 M NaCl), while Peak 2 eluted at about 76 ml (approximately 0.25 M NaCl). Peak 1 (DHP I) was selected for further purification. The purification procedure resulted in 31.1-fold purification and a homogeneous preparation of DHP I with recovery of about 25% of the starting enzyme activity ( Fig. 1and Table 1).
The pure DHP I native molecular weight was 30,790,
with a subunit molecular weight of 15,529. These data support a
holoenzyme composed of two identical subunits. The dehaloperoxidase is
a heme-containing protein, as indicated by the Soret absorbance peak at
420 nm and strong secondary peaks at 540 nm and 575 nm, and by
modification of this spectrum by reaction with pyridine (data not
shown). DHP I also contains 1 mol of iron per mol of dimer, as
determined by atomic absorption spectroscopy. This is consistent with a
content of one heme group per dimeric holoenzyme molecule. No other
metals were found in significant quantities. The amino acid composition
of the enzyme is given in Table 2with the composition of the N. lobatus chloroperoxidase provided for comparison. As is
typical of many peroxidases (see (9) ), both enzymes are rich
in aspartic acid (+ asparagine) and glutamic acid (+
glutamine). Interestingly, the sizes of the DHP I subunit (15,529) and
the chloroperoxidase subunit (15,500) are very similar and much
smaller than subunits of the many well characterized haloperoxidases
from plants, fungi, and bacteria(9) . The optimum pH for DHP I
activity is 5.0, which is also optimal for the phenol bromination
reaction catalyzed by the N. lobatus chloroperoxidase(9) . The trihalophenols
2,4,6-tribromophenol and 2,4,6-trichlorophenol are initially oxidized
to dihalogenated quinone intermediates (Fig. 2). Degradation of
these compounds continues to as yet unidentified products.
Figure 2: Mass spectra of major reaction products of the Amphitrite ornata dehaloperoxidase I using 2,4,6-tribromophenol (A) and 2,4,6-trichlorophenol (B) as substrates.
The A. ornata DHP I is very unusual in that it oxidizes a wide variety of halophenols, including mono-, di-, and trisubstituted halophenols with bromine, chlorine, or fluorine substituent groups (Table 3). This is particularly interesting given the stability of carbon-chlorine and carbon-fluorine bonds in haloaromatic compounds. DHP I is the first enzyme from a higher organism shown to oxidize these bonds at high specific activities and the first peroxidase capable of oxidizing carbon-fluorine bonds. As would be expected, bromophenols are oxidized at the highest rates of the halophenols tested. This is noteworthy since the great majority of known volatile halometabolites of infaunal worms are brominated aromatic compounds, and these are the compounds most abundant at sites inhabited by A. ornata(7) . The enzyme also shows little preference for either degree or position of substitution and can oxidize monohalogenated phenols at rates similar to those for di- and trihalogenated compounds.
DHP I represents approximately 3% of the soluble protein in crude extracts of A. ornata, an animal which lacks detectable haloperoxidase activity. DHP II is present at a roughly equivalent level. The other two polychaetes in which we have detected dehalogenase activity, Thelepus crispus and N. lobatus (Table 4), both produce halometabolites which are released into surrounding sediments(9, 13, 15) . In those species, dehalogenase activity is consistent with a role in prevention of autotoxicity. Since A. ornata produces no detectable volatile halometabolites and lacks detectable halogenase activity, autotoxicity seems unlikely. It lives, however, in close proximity to N. lobatus and S. kowalewskyi which contaminate the sediments with bromophenols and bromopyrroles(8, 13, 21) . A. ornata feeds on surface deposits with its tentacles and lives in a mudlined tube; its lifestyle therefore involves intimate contact with the sediments and associated contaminants(24) . The range of toxic bromoaromatic compounds encountered by A. ornata requires a detoxification mechanism which can neutralize high levels of several haloaromatic compounds having different structures and degrees and positions of bromine substitution. Sediments contaminated by anthropogenic sources, such as sediments downstream from sulfate process pulp mills, can also contain high levels of a diversity of haloaromatic compounds including chlorophenols, chloroguaiacols, and chlorocatechols, all potentially toxic to sediment-dwelling invertebrates(2, 25) . The high rates of A. ornata DHP I activity and its broad substrate specificity are consistent with its proposed function in neutralization of environmental haloaromatic toxins. We hypothesize that production of dehaloperoxidases allows A. ornata to survive in locations contaminated with these toxic compounds and that similar animals inhabiting sediments contaminated with haloaromatic compounds from both anthropogenic and biogenic sources will also produce these unusual dehaloperoxidases. Further structural and catalytic investigations of the A. ornata DHP I are ongoing.