A Role for Iron in an Ancient Carbonic Anhydrase - The Journal of ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 279, No. 8, Issue of February 20, pp. 6683–6687, 2004 Printed in U.S.A.

A Role for Iron in an Ancient Carbonic Anhydrase* Received for publication, October 23, 2003, and in revised form, November 29, 2003 Published, JBC Papers in Press, December 7, 2003, DOI 10.1074/jbc.M311648200

Brian C. Tripp‡§¶, Caleb B. Bell III‡¶, Francisco Cruz‡, Carsten Krebs‡, and James G. Ferry‡储 From the ‡Department of Biochemistry and Molecular Biology, Eberly College of Science, The Pennsylvania State University, University Park, Pennsylvania 16802-4500 and the §Departments of Biological Sciences and Chemistry, Western Michigan University, Kalamazoo, Michigan 49008

Since 1933, carbonic anhydrase research has focused on enzymes from mammals (␣ class) and plants (␤ class); however, two additional classes (␥ and ␦) were discovered recently. Cam, from the procaryote Methanosarcina thermophila, is the prototype of the ␥ class and the first carbonic anhydrase to be characterized from either an anaerobic organism or the Archaea domain. All of the enzymes characterized from the four classes have been purified aerobically and are reported to contain a catalytic zinc. Herein, we report the anaerobic reconstitution of apo-Cam with Fe2ⴙ, which yielded Cam with an effective kcat that exceeded that for the Zn2ⴙ-reconstituted enzyme. Mo¨ssbauer spectroscopy showed that the Fe2ⴙ-reconstituted enzyme contained high spin Fe2ⴙ that, when oxidized to Fe3ⴙ, inactivated the enzyme. Reconstitution with Fe3ⴙ was unsuccessful. Reconstitution with Cu2ⴙ, Mn2ⴙ, Ni2ⴙ, or Cd2ⴙ yielded enzymes with effective kcat values that were 10% or less than the value for the Zn2ⴙ-reconstituted Cam. Cam produced in Escherichia coli and purified anaerobically contained iron with effective kcat and kcat/Km values exceeding the values for Zn2ⴙ-reconstituted Cam. The results identify a previously unrecognized biological function for iron.

Carbonic anhydrase (CA),1 catalyzing the reversible hydration of CO2 to bicarbonate as shown in the equation below, was first purified from blood and characterized in 1933 (1). CO2 ⫹ H2O ⫽ HCO3 ⫹ H⫹

(Eq. 1)

Research in the intervening years has shown that CA is one of the most widely distributed enzymes in nature (2, 3) and continues to be intensely investigated. Amino acid sequence comparisons identify four classes (␣, ␤, ␥, and ␦) of independent origins (4). Isozymes of the ␣ class are found in virtually all mammalian tissues where they function in diverse essential processes. The ␤ class is ubiquitous in plants and algae, where it is indispensable for the acquisition and concentration of CO2 for photosynthesis. CA plays a role in the sequestration of atmospheric CO2 in carbonates, and the global cycles of silicon

* This work was supported by National Institutes of Health Grant GM44661 (to J. G. F.), National Science Foundation Training Grant Fellowship DBI-9602232 (to B. C. T.), NASA-Ames Research Laboratory Cooperative Agreement NCC21057 (to J. G. F.), and Funds from the Eberly College of Science at the Pennsylvania State University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ¶ These authors contributed equally to this work. 储 To whom correspondence should be addressed. Tel.: 814-863-5721; Fax: 814-863-6217; E-mail: [email protected]. 1 The abbreviations used are: CA, carbonic anhydrase; MOPS, 4-morpholinepropanesulfonic acid. This paper is available on line at http://www.jbc.org

and carbon are linked by CA in diatoms (5); thus, CA plays an important role in major geochemical and atmospheric processes. Members of the ␤ and ␥ classes are wide spread in physiologically diverse procaryotes from both the Bacteria and Archaea domains. Indeed, the genome of Escherichia coli contains two ␥ class homologs and two ␤ class homologs (2). Cam, from the procaryote Methanosarcina thermophila, is the prototype of the ␥ class and the first CA to be characterized from either an anaerobic organism or the Archaea domain (6). Sequence analyses approximate the evolution of the ␥ class at the estimated time of the origin of life (2). The crystal structure of Cam purified aerobically from E. coli reveals a homotrimer with a subunit fold composed of a left-handed ␤-helix motif followed by short and long ␣-helix structures (7). Each of the three active sites contain three histidines that coordinate a zinc ion. Two of the metal-binding histidines are donated by one monomer, and the third histidine from an adjacent monomer. Other residues in the active site of Cam are also donated from adjacent monomer faces and bear no resemblance to residues in the active site of the well characterized ␣ class CAs for which specific functions have been assigned. Kinetic investigations of the ␣ class CAs reveal a “zinc hydroxide” mechanism for catalysis (8) that also extends to both the ␤ and ␥ classes (9, 10). The overall enzyme-catalyzed reaction occurs in two mechanistically distinct steps, E-Zn2⫹-OH⫺ ⫹ CO2 ^ E-Zn2⫹-HCO3⫺

(Eq. 2)

E-Zn2⫹-HCO3⫺ ⫹ H2O ^ E-Zn2⫹-H2O⫹HCO3⫺

(Eq. 3)

E-Zn2⫹-H2O ^ ⫹H-E-Zn2⫹-OH⫺

(Eq. 4)

H-E-Zn2⫹-OH⫺ ⫹ B ^ E-Zn2⫹-OH⫺ ⫹ BH⫹

(Eq. 5)

⫹

where E is enzyme, and B is buffer. The first step is the interconversion between CO2 and bicarbonate (Equations 2 and 3) involving a nucleophilic attack of the zinc-bound hydroxyl on the CO2 molecule. The second step is regeneration of the zinc-bound hydroxide, which involves intramolecular proton transfer from the zinc-bound water to a proton shuttle residue (Equation 4) and intermolecular proton transfer to an accepting buffer molecule in the surrounding media (Equation 5). Kinetic analyses of site-specific replacement variants of Cam have identified essential residues (11, 12). All of the biochemically characterized CAs from the ␣, ␤, and ␥ classes are categorically described as zinc metalloenzymes; however, there are few reports investigating the role of metals other than zinc. Although in vitro replacement of zinc with cobalt in a few purified CAs from the ␣ and ␥ classes yields robustly active enzymes (10, 13, 14), it is unknown whether any cobalt-containing CAs are synthesized in vivo. A CA from the marine diatom Thalassiosira weissflogii contains cadmium (15), although there are no reports of a biochemical or structural characterization of the purified enzyme. In most cases,

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Iron-containing Carbonic Anhydrase

CAs are hyperproduced in E. coli cultured in medium supplemented with Zn2⫹ and purified aerobically with buffers containing Zn2⫹. Clearly, these procedures have the potential to bias the incorporation of Zn2⫹ in vivo during synthesis of the enzyme or by in vitro exchange of another metal for Zn2⫹ during purification. In particular, a role for iron in any CA has not been adequately investigated. Herein we show that replacement of zinc in Cam with ferrous iron yields an enzyme with effective kcat values exceeding zinc-containing Cam, identifying a previously unrecognized biological function for iron. EXPERIMENTAL PROCEDURES

Enzyme Purification—Hyperproduction of Cam in E. coli BL21(DE) was as described (16) except where indicated the growth medium was supplemented with either 0.01% ferric ammonium citrate or 0.5 mM ZnSO4. Cam was purified as described (11) except, where indicated, anaerobically utilizing an inert atmosphere glove bag (Coy Laboratory Products, Ann Arbor, MI) and buffers that were rendered O2-free by vacuum degassing and replacement with N2. After the final purification step, the fractions containing Cam were pooled, dialyzed against 50 mM MOPS (pH 7.5), placed in aliquots, frozen in liquid N2, and stored at ⫺80 °C until further use. When purified anaerobically, no metals were added to the buffers, and all solutions were treated with Chelex-100 resin (Bio-Rad). In addition, all of the glassware was acid-washed and rinsed with Chelex-treated water. Preparation of apo-Cam and Metal Reconstitution—In contrast to ␣ class carbonic anhydrases (14), the Cam active site metal cannot be readily removed by dialysis with chelators such as dipicolinate. Thus, Cam (10 –50 mg/ml) was unfolded to allow removal of existing metals by chelation and then refolded to produce apo-Cam that was subsequently reconstituted with the indicated metals. Purified Cam (10 –50 mg/ml) was incubated in 50-ml polyethylene centrifuge tubes at 4 °C for 16 h in 20 mM MOPS (pH 7.5) containing 4.0 M spectroscopic grade guanidine hydrochloride (Fisher) and 50 mM dipicolinate. All of the buffers were treated with Chelex-100 resin to remove transition metal ions. The metal-free unfolded protein was then transferred into metal-free Spectra/Por 7 MWCO8000 tubing (Spectrum Laboratories, Rancho Dominguez, CA) and dialyzed three times for 24 h at 4 °C against 25 mM MOPS (pH 7.5) containing 150 mM KCl, 25 mM dipicolinate, and 1 mM dithiothreitol. The dipicolinate and dithiothreitol were then removed from the refolded apo-Cam by 3⫻ dialysis against 25 mM MOPS (pH 7.5) containing 150 mM KCl for 24 h at 4 °C. The apo-Cam was reconstituted with the indicated metals by dialysis twice for 24 h with 25 mM MOPS (pH 7.5) containing 150 mM KCl and either 200 ␮M ZnSO4, 200 ␮M CdSO4, 200 ␮M CuSO4, 200 ␮M FeSO4, 200 ␮M FeCl3, 2 mM CoSO4, 2 mM NiSO4, 2 mM MgSO4, or 2 mM MnSO4. A competitive dialysis experiment was also performed anaerobically using 200 ␮M CoSO4, 200 ␮M FeSO4, and 200 ␮M ZnSO4. The reconstituted enzymes were subjected to size exclusion chromatography with a Superose 75 (Amersham Biosciences) column developed with Chelex-treated 25 mM MOPS (pH 7.5) containing 150 mM KCl to remove misfolded protein aggregates. Reconstitutions of apo-Cam with FeSO4 and size exclusion chromatography of the reconstituted enzymes were performed in the anaerobic glove bag at 25 °C. The reconstituted enzymes were placed in aliquots, frozen in liquid nitrogen, and stored at ⫺80 °C. Analytical—Cam preparations were analyzed for metals (aluminum, boron, barium, calcium, cadmium, cobalt, chromium, copper, iron, potassium, magnesium, manganese, molybdenum, sodium, nickel, phosphorus, lead, silicon, strontium, and zinc) by inductively coupled plasma emission spectrophotometry using a Jarrell-Ash 965 ICP instrument at the Chemical Analysis Laboratory of the University of Georgia. Circular dichroism spectra were collected on an AVIV model 62 DS spectrophotometer (Lakewood, NJ) at 25 °C using a 2-mm path length quartz cuvette and a monomer concentration of 10 ␮M in 50 mM, MOPS (pH 7.5) containing 50 mM Na2SO4. The protein solutions were scanned over 200 –260 nm using a bandwidth of 2.0 nm and a data acquisition time of 5 s. The Fe2⫹ content of Cam was determined using the ferrouschelating chromophore ferrozine (3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-trizine) as previously described (17) except the assay mixture also contained 6.1 M guanidine hydrochloride to unfold the protein and allow the otherwise inaccessible ligated Fe2⫹ to bind ferrozine. Carbon dioxide hydrating activity was measured by stopped flow spectroscopy using the changing pH indicator method as described previously (10), except for enzymes purified anaerobically, in which case all of the solutions were anaerobic. The protein concentration determinations were performed as described (10). The Mo¨ ssbauer spectra were

recorded on a spectrometer (WEB Research, Edina, MN) operating in the constant acceleration mode in a transmission geometry. During the measurement the sample was kept inside a SVT-400 Dewar from Janis (Wilmington, MA) at a temperature of 4.2 K in a magnetic field of 40 milliteslas applied parallel to the ␥-beam. Data analysis was performed using the program WMOSS (WEB Research). RESULTS

Unfolding Cam with denaturant in the presence of a metal chelator and subsequent refolding by removal of the denaturant yield trimeric apo-Cam that can be reconstituted with nearly a full complement of Zn2⫹ or Co2⫹ producing highly active enzymes (10). The crystal structures of Zn2⫹- and Co2⫹reconstituted Cam show the metals coordinated to the nitrogen atoms of histidines in all three active sites (18). The properties of Cam reconstituted with Zn2⫹, Co2⫹, and several other metal ions are shown in Table I. Incorporation of Zn2⫹ or Fe2⫹ was nearly quantitative with respect to the three available metal binding sites. Although substoichiometric, reconstitution was substantial for Co2⫹, Cu2⫹, Mn2⫹, Ni2⫹, and Cd2⫹. Analysis for a suite of metals showed that the sum of contaminating metals (other than the indicated reconstitution metal) was less than 0.18/trimer for each reconstituted enzyme. An exception was the 57Fe2⫹-reconstituted Cam, for which a significant amount of contaminating zinc was incorporated. Attempts to reconstitute with Fe3⫹ or Mg2⫹ yielded enzymes with negligible amounts of the respective metals. The coordination of metal by histidines from adjacent subunits contributes to the stability of the trimer (11); thus, apoCam is susceptible to subunit dissociation during size exclusion column chromatography. Recovery of the trimeric form of the reconstituted enzymes from the size exclusion column was proportional to the metal content (data not shown), a result indicating that the metals were coordinated in the active site. Attempts to reconstitute apo-Cam with Fe3⫹ or Mg2⫹ resulted in a nearly complete loss of the trimeric form consistent with the metal content, indicating that the active site of Cam is unable to accommodate these metals. The effective kcat and kcat/Km values (Table I) for the Zn2⫹and Co2⫹-reconstituted enzymes were similar to published values (10). Notably, the Fe2⫹-reconstituted Cam had effective kcat values that exceeded those for the Zn2⫹-and Co2⫹-reconstituted enzymes. Circular dichroism spectra for the Zn2⫹- and Fe2⫹reconstituted enzymes were nearly identical, indicating a similar secondary structure (Fig. 1). These results indicate that iron functions at least as well as zinc in catalysis and also for the stability of the trimer. The divalent metals Cu2⫹, Mn2⫹, Ni2⫹, and Cd2⫹ also replaced Zn2⫹; however, the low effective kcat values of these reconstituted enzymes relative to the value for the Fe2⫹-reconstituted Cam suggest that they are not physiologically relevant. The ferrozine assay indicated that iron present in the Fe2⫹reconstituted enzyme was ferrous. Exposure to air diminished the kcat with a half-life of less than 5 min (Fig. 2), indicating the Fe2⫹-reconstituted Cam is oxygen-sensitive. The Mo¨ ssbauer spectrum of 57Fe2⫹-reconstituted Cam is shown in Fig. 3B. The spectrum has two broad absorption lines at ⫺0.5 mm/s and ⫹2.9 mm/s. The spectrum collected over a narrower range of Doppler velocities (Fig. 3A) reveals the presence of at least two overlapping quadrupole doublets. The solid line overlaid with the experimental data in Fig. 3A is a theoretical simulation assuming two quadrupole doublets having the following parameters: isomer shift ␦1 ⫽ 1.19 ⫾ 0.03 mm/s, quadrupole splitting parameters ⌬EQ1 ⫽ 3.00 ⫾ 0.03 mm/s (47% of relative intensity); and ␦2 ⫽ 1.15 ⫾ 0.03 mm/s, ⌬EQ2 ⫽ 3.54 ⫾ 0.03 mm/s (53% of relative intensity). These parameters are typical of high spin Fe2⫹ sites coordinated by five or six nitrogen/ oxygen ligands. The occurrence of two (or more) different iron

Iron-containing Carbonic Anhydrase

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TABLE I Metal content and kinetic constants of Cam preparations Growth supplementa

Purification

Aerobic

Anaerobic

Reconstitution metal

Zn2⫹ Zn2⫹ Zn2⫹ Zn2⫹ Zn2⫹ Zn2⫹ Zn2⫹ Zn2⫹ Fe3⫹ Fe3⫹ Fe3⫹

Zn2⫹ Co2⫹ Cu2⫹ Mn2⫹ Ni2⫹ Cd2⫹ Mg2⫹ Fe3⫹ Fe2⫹ 57 Fe2⫹ Zn2⫹,Fe2⫹,Co2⫹

Fe3⫹

No reconstitution

Zn2⫹

No reconstitution

No supplementation

No reconstitution

Molar ratio of metal/trimer

Effective kcat (⫻10⫺3 s⫺1)

Km (⫻103 M)

Effective kcat/Km (⫻10⫺5 M⫺1 s⫺1)

2.76 ⫾ 0.15 zinc 2.37 ⫾ 0.09 cobalt 1.47 ⫾ 0.06 copper 2.34 ⫾ 0.09 manganese 2.37 ⫾ 0.09 nickel 2.46 ⫾ 0.12 cadmium ⬍ 0.01 manganesec ⬍ 0.01 ironc 2.79 ⫾ 0.12 iron 2.25 ⫾ 0.12 iron 0.69 ⫾ 0.06 zinc 2.37 ⫾ 0.03 zinc 0.33 ⫾ 0.03 iron 0.21 ⫾ 0.03 cobalt 2.70 ⫾ 0.12 iron 0.30 ⫾ 0.03 zinc 1.17 ⫾ 0.03 zinc 1.02 ⫾ 0.03 iron 1.89 ⫾ 0.03 iron 0.27 ⫾ 0.03 inc 0.04 ⫾ 0.01 copper

68.1 ⫾ 4.0b 118.0 ⫾ 10.5b 9.2 ⫾ 0.4b 5.8 ⫾ 0.5b 0.7 ⫾ 0.1b 5.5 ⫾ 0.3b NDd ND 243.3 ⫾ 22.8b 110.2 ⫾ 9.6e 55.1 ⫾ 7.7e

21.8 ⫾ 2.2 15.7 ⫾ 2.8 7.2 ⫾ 0.8 22.5 ⫾ 3.4 14.2 ⫾ 2.8 22.1 ⫾ 2.0 ND ND 44.8 ⫾ 7.4 24.6 ⫾ 3.5 24.3 ⫾ 5.7

31.3 ⫾ 5.1 75.3 ⫾ 19.9 12.9 ⫾ 1.9 2.6 ⫾ 0.6 0.5 ⫾ 0.1 2.5 ⫾ 0.4 ND ND 54.4 ⫾ 15.5 44.8 ⫾ 10.4 22.7 ⫾ 8.6

206.4 ⫾ 61.7e

26.1 ⫾ 9.3

79.0 ⫾ 51.8

190.1 ⫾ 44.4e

40.7 ⫾ 13.3

46.7 ⫾ 26.2

275.1 ⫾ 7.7

46.0 ⫾ 7.8

59.9 ⫾ 17.5

e

a

The enzymes were purified from E. coli cultured in medium supplemented with either 0.01 % (w/v) ferric ammonium citrate or 0.5 mM ZnSO4. Effective kcat and kcat/Km values were obtained by dividing apparent kcat and kcat/Km values by the molar ratio of metal/monomer. Limit of detection. d ND, not determined. e Effective kcat and kcat/Km values were obtained by dividing apparent kcat and kcat/Km values by the sum of the molar ratio of metals/monomer. b c

FIG. 1. Circular dichroism spectra of Cam. Solid line, Zn2⫹-reconstituted; dashed line, Fe2⫹-reconstituted.

species may reflect binding of different ligands (e.g. OH⫺, H2O, or HCO3⫺) to the iron center. Exposure of Zn2⫹-Cam to H2O2 had little effect on activity; however, the same treatment of Fe2⫹-reconstituted Cam rapidly produced a brown color with vigorous bubbling and reduced the kcat value to less than 10% consistent with oxidation of Fe2⫹ to Fe3⫹ (Fig. 4). The Mo¨ ssbauer spectrum of H2O2-inactivated 57Fe2⫹-reconstituted Cam (Fig. 3C) exhibited a broad, magnetically split, six-line spectrum that is typical of high spin Fe3⫹ in the slow relaxation limit, which unambiguously demonstrates that exposure to H2O2 results in complete (⬎98%) oxidation of the high spin Fe2⫹ center. These results establish that the enzyme contains high spin ferrous iron and that oxidation to ferric iron inactivates the enzyme. The residual activity after oxidation of 57 Fe2⫹-Cam is attributed to zinc-Cam (Table I) produced by reconstitution of apo-Cam with zinc contaminating the 57Fe2⫹. Anaerobic size exclusion chromatography of air-oxidized Fe2⫹-Cam showed nearly complete loss of the trimeric form (not shown), indicating subunit dissociation as a consequence of the loss of Fe3⫹ from the active site. These results are consistent with the inability of Fe3⫹ to bind to apo-Cam (Table I) and suggest that only Fe2⫹ is able to coordinate in the active

FIG. 2. Air inactivation of Fe2ⴙ-reconstituted Cam. Samples of the enzyme (Table I) were assayed by the stopped flow method using metal-free buffers at the times indicated after exposure to 1.0 atmosphere of air at 25 °C. A control incubated anaerobically retained 100% activity over the time period shown (data not shown).

site. These results also suggest that Fe2⫹-Cam synthesized in vivo could lose iron during aerobic purification because of oxidation of Fe2⫹ to Fe3⫹ with oxygen. All published descriptions of unreconstituted Cam report zinc as the only metal; however, the enzyme was produced in E. coli cultured in medium supplemented with Zn2⫹ and purified aerobically with buffers amended with Zn2⫹ (6, 16). The low yields obtained both for cell mass and purified enzyme have precluded a reliable metal analysis of Cam produced in M. thermophila; thus, Cam was produced in E. coli that was cultured in unsupplemented medium or media supplemented with either Fe2⫹, Zn2⫹, or both and purified anaerobically without supplementation of buffers with metals. Table I shows the metal content and kinetic parameters. Each of the unreconstituted Cam preparations had effective kcat and kcat/Km values that approximated those for Fe2⫹-reconstituted Cam, a result consistent with the metal

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Iron-containing Carbonic Anhydrase that E. coli produced predominantly Zn2⫹-Cam, and the Zn2⫹ was exchanged for Fe2⫹ during anaerobic purification. These results establish that E. coli synthesizes Fe2⫹-Cam in vivo. Remarkably, Cam from E. coli cells cultured in medium supplemented with only Zn2⫹ (0.5 mM) contained iron in amounts approximately equal to that of zinc (Table I). This result is in stark contrast to that predicted by the IrvingWilliams series, for which the stability of complexed Fe2⫹ is at least an order of magnitude less than that for Zn2⫹, Co2⫹, and Cu2⫹ ligated with nitrogen (19); indeed, anaerobic reconstitution of apo-Cam with equimolar amounts of Fe2⫹, Zn2⫹, and Co2⫹ produced enzyme with predominantly zinc (Table I). Furthermore, E. coli cultured in unsupplemented medium produced Cam with 7-fold more iron than zinc, although metals analysis of the medium showed it contained 1.6 ppm of iron and 3.2 ppm of zinc. Finally, Cam purified from cells cultured in medium supplemented with only Zn2⫹ contained substoichiometric amounts of iron plus zinc in contrast to Cam purified from cells cultured in medium supplemented with Fe2⫹. These results establish that E. coli incorporates Fe2⫹ into Cam with greater efficiency compared with Zn2⫹. DISCUSSION

FIG. 3. Mo¨ ssbauer spectra of 57Fe2ⴙ-reconstituted Cam. Spectra A and B, no treatment; spectrum C, H2O2-inactivated. The solid line overlaid with the experimental data for A is a theoretical simulation using the parameters quoted in the text. The Doppler velocity scale of A is shown at the top, and the scale for B and C is shown at the bottom.

FIG. 4. Hydrogen peroxide inactivation of 57Fe2ⴙ-reconstituted and Zn2ⴙ-reconstituted Cam. Hydrogen peroxide (final concentration, 3%) was added to the Cam samples (0.4 ␮M), incubated at 25 °C for 10 min, and assayed immediately. The 100% activity (effective kcat values) for each of the reconstituted enzymes are shown in Table I.

analysis showing incorporation of Fe2⫹. The sum of metals other than iron or zinc for each growth condition was less than 0.03/trimer, except for the enzyme purified from cells cultured in unsupplemented medium, which contained copper albeit in much lower amounts than iron or zinc (Table I). The enzyme purified from cells cultured in unsupplemented medium contained less than 0.01 cobalt/trimer. Substoichiometric amounts of metals were present in the enzymes purified from E. coli cultured in Zn2⫹-supplemented and unsupplemented media, indicating the presence of apo-protein in both of these enzyme preparations, results suggesting that iron and zinc were limiting for both culture conditions. The iron content of the enzyme purified from cells cultured in Fe2⫹-supplemented medium exceeded the zinc content by 9-fold. In contrast to the ␣ class CAs (14), the Cam active site metal cannot be readily removed by dialysis with metal chelators unless the enzyme is unfolded with a chemical denaturant (10, 11). Thus, it is highly unlikely

The results presented here establish a previously unrecognized biological role for iron. It is reported that Cam from M. thermophila aerobically purified from E. coli cells cultured in Zn2⫹-supplemented medium contains substoichiometric amounts of zinc and no appreciable amounts of other metals, which lead to the conclusion that Cam is a zinc metalloenzyme (16). However, as reported here, Cam contained approximately equal amounts of iron and zinc when purified anaerobically from E. coli cultured in medium supplemented with Zn2⫹. Based on the results reported here, it is likely that the previously reported Zn2⫹-Cam (16) also contained Fe2⫹ in vivo that was oxidized to Fe3⫹ and lost from the enzyme during aerobic purification. This result is not without precedent. Peptide deformylase from E. coli was first reported to contain Zn2⫹ when purified aerobically and later shown to utilize Fe2⫹ for catalysis (20). Oxygen inactivates the deformylase by oxidation of Fe2⫹ to Fe3⫹, and exclusion of oxygen favors the incorporation of Fe2⫹ over Zn2⫹ (21). Use of strictly anaerobic conditions to prevent oxidation also lead to the identification of Fe2⫹ as the active site metal in the previously reported Zn2⫹-containing methionyl aminopeptidase from E. coli (22). Homologs of Cam genes are present in the genome of E. coli (YrdA and CaiE) consistent with synthesis of Cam homologs in this organism. The finding that E. coli synthesizes Fe2⫹-Cam with higher efficiency relative to Zn2⫹-Cam establishes that Fe2⫹-Cam is the predominant in vivo metal form. Furthermore, the results suggest that E. coli synthesizes Fe2⫹-Cam in nature. This proposition is supported by the preponderance of Fe2⫹-Cam synthesized by cells cultured in unsupplemented medium that more closely mimic conditions in the native environment. Furthermore, unsupplemented medium contained twice the amount of zinc relative to iron, and the Irving-Williams series predicts a greater affinity for Zn2⫹ over Fe2⫹, as was shown for anaerobic reconstitution of apo-Cam with competing amounts of these metals. Other considerations argue for the synthesis of Fe2⫹-Cam by a diversity of anaerobes in their native anoxic environments. In addition to M. thermophila and E. coli, homologs of the gene encoding Cam are found in many strict and facultative anaerobes (2) that proliferate in anoxic environments where Fe2⫹ availability is severalfold greater than Zn2⫹ (23). Indeed, the metabolic pathways of anaerobes including M. thermophila (24) evolved to utilize an abundance of diverse oxygen-sensitive Fe2⫹-containing proteins (25). Finally, sequence analyses approximates the origin for the ␥ class

Iron-containing Carbonic Anhydrase (2) to the time when Fe2⫹-containing proteins are thought to have played a major role in the origin and early evolution of life in an anoxic and Fe2⫹-rich aqueous environment (26, 27). Although reconstitution of apo-Cam with Co2⫹ yielded an enzyme with a catalytic efficiency between Fe2⫹- and Zn2⫹-reconstituted Cam (10), the significant synthesis of Co2⫹-Cam in nature is unlikely. This proposition is supported by the results showing that cobalt was negligible in unreconstituted Cam that was anaerobically purified from cells cultured in unsupplemented medium. Finally, the intracellular concentration of cobalt reported for M. thermophila is 32-fold less than for iron, reflecting the relative availability of these metals in the native environment (28). It was found that Fe2⫹ is incorporated in vivo with a greater efficiency than Zn2⫹ in contrast to that predicted by the IrvingWilliams series; however, the mechanism by which E. coli preferentially incorporates Fe2⫹ is unknown. If incorporation is by unassisted binding of uncomplexed metals, then the IrvingWilliams series requires a concentration of free Fe2⫹ severalfold greater than Zn2⫹ in the cytosol. This scenario is supported by the report that free Zn2⫹ in the E. coli cytosol is less than one atom/cell (29). Furthermore, Cam purified anaerobically from cells cultured in Zn2⫹-supplemented medium contained significant amounts of apo-Cam, suggesting that the cells were limited for Zn2⫹, whereas Cam purified anaerobically from cells cultured in Fe2⫹-supplemented medium contained a nearly full complement of iron. On the other hand, if the intercellular concentrations of free Fe2⫹ and Zn2⫹ are comparable, it is likely that a chaperonin assists Fe2⫹ incorporation into Cam. The presence of genes in E. coli with homology to the Cam gene from M. thermophila presents the possibility of a chaperonin in E. coli that also functions to incorporate Fe2⫹ in Cam. The results reported here raise a question regarding the extent to which Fe2⫹ functions in CAs from other classes. There are no previous reports of the anaerobic purification or assay of any CA, and most often Zn2⫹ is included in buffers used for aerobic purification. The anaerobic purification of CA from bovine erythrocytes yields an enzyme with 0.95 atoms of zinc,2 suggesting that Fe2⫹-containing ␣ class CAs are inconsequential in mammalian cells. These results are not surprising considering the low abundance of Fe2⫹ in mammalian tissue. However, the report that CAs are up-regulated in anoxic cancer cells (30) warrants an investigation of the metal content for these enzymes. Clearly, the results reported here call for a re-evaluation of the role of Fe2⫹ in isozymes from all classes of CA utilizing anaerobic methods to exclude oxidation of Fe2⫹. Indeed, CAs from the ␤ class are found in strictly anaerobic 2

C. B. Bell III and J. G. Ferry, unpublished data.

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microbes (2, 31, 32) that proliferate in Fe2⫹-rich environments and are highly dependent on Fe2⫹-containing proteins. Clarification of the in vivo metal form in CAs will have a profound influence on the biological understanding of this ubiquitous enzyme. The results also raise a caution for other enzymes characterized under aerobic conditions and reported to utilize zinc. Acknowledgments—We thank Brandon Doyle and C. Robert Matthews for helpful advice, discussions, and training on refolding of Cam and use of the circular dichroism spectrometer. We also thank Andrea Gorrell and Sarah Lawrence for critical reading of the manuscript. REFERENCES 1. Meldrum, N. N., and Roughton, F. J. W. (1933) J. Physiol. 80, 113–142 2. Smith, K. S., Jakubzick, C., Whittam, T. C., and Ferry, J. G. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 15184 –15189 3. Hewett-Emmett, D. (2000) in The Carbonic Anhydrases (Chegwidden, W. R., Carter, N. D., and Edwards, Y. H., eds) pp. 29 –76, Birkhauser Verlag, Basel, Switzerland 4. Tripp, B. C., Smith, K., and Ferry, J. G. (2001) J. Biol. Chem. 276, 48615– 48618 5. Milligan, A. J., and Morel, F. M. (2002) Science 297, 1848 –1850 6. Alber, B. E., and Ferry, J. G. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 6909 – 6913 7. Kisker, C., Schindelin, H., Alber, B. E., Ferry, J. G., and Rees, D. C. (1996) EMBO J. 15, 2323–2330 8. Lindskog, S. (1997) Pharmacol. Ther. 74, 1–20 9. Rowlett, R. S., Chance, M. R., Wirt, M. D., Sidelinger, D. E., Royal, J. R., Woodroffe, M., Wang, Y. F. A., Saha, R. P., and Lam, M. G. (1994) Biochemistry 33, 13967–13976 10. Alber, B. E., Colangelo, C. M., Dong, J., Stålhandske, C. M. V., Baird, T. T., Tu, C., Fierke, C. A., Silverman, D. N., Scott, R. A., and Ferry, J. G. (1999) Biochemistry 38, 13119 –13128 11. Tripp, B. C., Tu, C., and Ferry, J. G. (2002) Biochemistry 41, 669 – 678 12. Tripp, B. C., and Ferry, J. G. (2000) Biochemistry 39, 9232–9240 13. Lindskog, S. (1966) Biochemistry 5, 2641–2646 14. Hunt, J. A., Ahmed, M., and Fierke, C. A. (1999) Biochemistry 38, 9054 –9062 15. Lane, T. W., and Morel, F. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 4627– 4631 16. Alber, B. E., and Ferry, J. G. (1996) J. Bacteriol. 178, 3270 –3274 17. Dailey, H. A., Jr., and Lascelles, J. (1977) J. Bacteriol. 129, 815– 820 18. Iverson, T. M., Alber, B. E., Kisker, C., Ferry, J. G., and Rees, D. C. (2000) Biochemistry 39, 9222–9231 19. Sigel, H., and McCormick, D. B. (1970) Acc. Chem. Res. 3, 201–208 20. Rajagopalan, P. T., Yu, C. X., and Pei, D. (1997) J. Am. Chem. Soc. 119, 12418 –12419 21. Rajagopalan, P. T., and Pei, D. (1998) J. Biol. Chem. 273, 22305–22310 22. D’Souza V, M., and Holz, R. C. (1999) Biochemistry 38, 11079 –11085 23. Frausto da Silva, J. J. R., and Williams, R. J. P. (2001) Biological Chemistry of the Elements: The Inorganic Chemistry of Life, 2nd Ed., Oxford University Press, New York 24. Ferry, J. G. (1999) FEMS Microbiol. Rev. 23, 13–38 25. Beinert, H., Holm, R. H., and Munck, E. (1997) Science 277, 653– 659 26. Wachtershauser, G. (1990) Origins Life Evolution Biosphere 20, 173–176 27. Johnson, M. K. (1998) Curr. Opin. Chem. Biol. 2, 173–181 28. Scherer, P., Lippert, H., and Wolff, G. (1983) Biol. Trace Element Res. 5, 149 –163 29. Outten, C. E., and O’Halloran, T. V. (2001) Science 292, 2488 –2492 30. Potter, C. P., and Harris, A. L. (2003) Br. J. Cancer 89, 2–7 31. Strop, P., Smith, K. S., Iverson, T. M., Ferry, J. G., and Rees, D. C. (2001) J. Biol. Chem. 276, 10299 –10305 32. Smith, K. S., and Ferry, J. G. (2000) FEMS Microbiol. Rev. 24, 335–366