(Received for publication, September 6, 1995; and in revised form, January 23, 1996)
From the
The selenocysteine-containing formate dehydrogenase H (FDH) is
an 80-kDa component of the Escherichia coli formate-hydrogen
lyase complex. The molybdenum-coordinated selenocysteine is essential
for catalytic activity of the native enzyme. FDH in dilute solutions
(30 µg/ml) was rapidly inactivated at basic pH or in the presence
of formate under anaerobic conditions, but at higher enzyme
concentrations (3 mg/ml) the enzyme was relatively stable. The
formate-reduced enzyme was extremely sensitive to air inactivation
under all conditions examined. Active formate-reduced FDH was
crystallized under anaerobic conditions in the presence of ammonium
sulfate and PEG 400. The crystals diffract to 2.6 Å resolution
and belong to a space group of P4
2
2 or
P4
2
2 with unit cell dimensions a = b = 146.1 Å and c =
82.7 Å. There is one monomer of FDH per crystallographic
asymmetric unit. Similar diffraction quality crystals of oxidized FDH
could be obtained by oxidation of crystals of formate-reduced enzyme
with benzyl viologen. By EPR spectroscopy, a signal of a single reduced
FeS cluster was found in a crystal of reduced FDH, but not in a crystal
of oxidized enzyme, whereas Mo(V) signal was not detected in either
form of crystalline FDH. This suggests that Mo(IV)- and the reduced FeS
cluster-containing form of the enzyme was crystallized and this could
be converted into Mo(VI)- and oxidized FeS cluster form upon oxidation.
A procedure that combines anaerobic and cryocrystallography has been
developed that is generally applicable to crystallographic studies of
oxygen-sensitive enzymes. These data provide the first example of
crystallization of a substrate-reduced form of a Se- and Mo-containing
enzyme.
Molybdenum-containing enzymes are widely distributed in both eukaryotes and prokaryotes and have important functional roles in living organisms and in biogeochemical cycles(1, 2) . All known Mo-containing enzymes except nitrogenase (3) are members of a large family of proteins in which molybdenum is coordinated to a molybdopterin(4, 5) . In Escherichia coli formate dehydrogenase, one of two known selenium-dependent molybdoenzymes(6, 7) , molybdenum is also coordinated to selenium of a selenocysteine residue(7, 8) .
Although several molybdopterin-dependent enzymes have been studied extensively by mechanistic and structural methods for decades (9, 10) , there is still a lack of definitive information on reaction mechanisms and three-dimensional structures. X-ray structural analyses of some of these proteins have been hampered by microheterogeneity due to the partial loss of cofactors or their components, as well as by enzyme instability which usually resulted in failure in crystallization or in decreased diffraction of the crystals. The crystallization of two molybdopterin-containing enzymes, a native aldehyde oxidoreductase from Desulfovibrio gigas(11, 12) and a bovine xanthine oxidase, has been reported(13) .
Important information on molybdenum-containing enzymes perhaps can be inferred from studies on tungsten-containing enzymes. The structure of the tungstoenzyme, aldehyde ferredoxin oxidoreductase, from Pyrococcus furiosus was solved recently at 2.3-Å resolution (14) . The active center tungsten is not bound to amino acid residues of the polypeptide chain as suggested for certain bacterial molybdoenzymes (7) ; rather, tungsten is coordinated to two identical molecules of molybdopterin, each molybdopterin providing two thiolene sulfurs for tungsten coordination. By analogy, these studies supported the proposed structure of the molybdopterin molecule and the proposed type of metal-molybdopterin coordination. However, the archaebacterial aldehyde ferredoxin oxidoreductase does not have amino acid sequence homology to known molybdenum-containing enzymes.
Substrates of molybdoenzymes react with molybdenum active centers resulting in the redox transformations of these centers and the formation of unstable enzymic intermediates. Some of these are paramagnetic species which have been the subject of extensive studies by EPR, ENDOR (electron nuclear double resonance), and EXAFS (extended x-ray absorption fine structure) spectroscopies(2, 9) . However, the interpretation of the data obtained with these techniques depends largely upon our knowledge of three-dimensional structures, especially the structures of reduced forms of the enzymes.
Selenium- and
molybdenum-containing formate dehydrogenase H (FDH) ()and
hydrogenase 3 are components of the formate-hydrogen lyase complex,
which in vivo decomposes formic acid to carbon dioxide and
hydrogen under anaerobic conditions (15) . FDH was purified
recently (16, 17, 18) and was shown to
contain a number of redox centers: a molybdopterin cofactor consisting
of molybdenum coordinated to molybdopterin guanine
dinucleotide(16) , a selenocysteine (SeCys) residue (8) encoded by UGA(19) , and a FeS
cluster(16) . The catalytic activity of mutant enzyme in which
SeCys-140 was substituted with Cys was only 0.3% that of the wild type
enzyme(18) , and a Ser-140 mutant was inactive(20) .
EPR studies of formate-reduced Mo(V)-containing FDH have demonstrated
the direct coordination of selenium to molybdenum(7) . FDH is
extremely oxygen-sensitive, and isolation of the active enzyme can be
achieved only under strictly anaerobic conditions. In the presence of
substrate, formate, the enzyme is inactivated in a time-dependent
manner(16) .
In spite of its oxygen sensitivity, FDH may be a useful model for studies on molybdenum- and selenium-containing metalloenzymes. Since the enzyme is a monomer of 80 kDa and lacks flavin, it has certain advantages over more complex multicomponent systems. Due to the presence of selenium in the molybdenum coordination sphere, additional methods could be applied to investigate enzyme structure and reaction mechanism.
Herein we present an improved purification procedure, methods of stabilization, and crystallization of substrate-reduced enzyme, together with a procedure for oxidation of the enzyme in crystals and preliminary crystallographic analysis of formate dehydrogenase.
The procedure for isolation of FDH from 300 g of cells was essentially the same as described previously(16) , except that cells were ruptured by sonication and the isolation procedure was scaled up 15 times to obtain larger amounts of protein. The entire purification procedure was completed in 3-4 days. FDH obtained after the phenyl-Sepharose and hydroxyapatite chromatographic steps was more than 95% pure according to analytical gel filtration and SDS-PAGE analyses. 50 mg of enzyme was obtained with a specific activity of 950 µmol/min/mg of protein. To further purify the enzyme, an additional ion-exchange chromatographic step was designed, in which FDH in 25 mM MES/NaOH, pH 6.0, 3 mM sodium azide, was applied to a DEAE-HPLC column equilibrated with 25 mM sodium phosphate, pH 6.3, 3 mM sodium azide (buffer A). The enzyme was eluted with a linear gradient of 0 to 200 mM sodium sulfate in buffer A. The active enzyme fractions were pooled and made to 25 mM MES/NaOH, pH 6.0, 5 mM sodium azide, 1 mM sodium phosphate, 3 mM sodium sulfate by diafiltration on an Amicon ultrafiltration unit fitted with a YM-10 membrane. FDH was concentrated to 11 mg/ml, passed through a 0.22-µm filter and frozen by dripping into liquid nitrogen, then stored in liquid nitrogen. No loss of activity was detected in thawed samples. For experiments with FDH, droplets of the frozen enzyme were brought into an anaerobic chamber or the NIH Anaerobic Laboratory and thawed in anaerobic microcentrifuge tubes.
To test the effect of oxygen on stability of FDH, 50-µl
samples containing 150 µg, 15 µg, or 1.5 µg of FDH in 25
mM MES/NaOH, pH 6.0, and 3 mM sodium azide were
incubated for 2 h in closed 1.5-ml microcentrifuge tubes (gas phase
air) at room temperature. The samples then were brought into the NIH
Anaerobic Laboratory, flushed with argon, transferred into deoxygenated
microcentrifuge tubes, and incubated overnight (gas phase 99%
N, 1% H
) at 4 °C. Catalytic activities were
measured in the standard assays and compared to the activity of a
control sample (150 µg of FDH/50 µl), which was not exposed to
air and had been kept in the NIH Anaerobic Laboratory. These
experiments were performed both in the presence and in the absence of
20 mM sodium formate.
An additional ion-exchange chromatographic purification step separated some impurities from the main FDH peak (Fig. 1). The purity of the resulting FDH preparation was confirmed with native and SDS-PAGE, analytical gel filtration and mass spectrometry (MALDI) analyses (not shown). A molecular mass of about 80 kDa estimated with these techniques matched the molecular mass predicted from the FDH gene sequence (79.1 kDa), indicating the presence of a full-length polypeptide. The UV-visible absorption spectrum (Fig. 2) was characteristic of a protein containing iron-sulfur clusters with a 280 nm to 400 nm absorbance ratio of 9. Residual trace amounts of hydrogenase activity were not separated from formate dehydrogenase activity by the DEAE-chromatographic step and later by crystallization. The ratio of formate dehydrogenase specific activity to hydrogenase specific activity was 733 for the isolated enzyme and 933 for the enzyme obtained from dissolved crystals.
Figure 1: Purification of FDH on a DEAE-HPLC column. FDH purified by phenyl-Sepharose and hydroxyapatite chromatography was further purified on a DEAE-HPLC column as described under ``Experimental Procedures.'' Elution was followed by absorption measurements at 280 nm. The main peak eluted at 1570 s represents FDH. Minor peaks represent impurities.
Figure 2: Absorption spectra of FDH. The electronic absorption spectrum of ``as isolated'' FDH is shown (lower spectrum). The area where FeS clusters absorb is shown in the enlarged scale (upper spectrum). Inset shows the difference spectrum (oxidized enzyme minus formate reduced enzyme). The maximum in the difference spectrum is at 450 nm due to the reduction of FeS clusters. The enzyme was reduced with 10 mM formate for 5 min at room temperature.
Figure 3:
Stability of FDH: effect of pH, sodium
formate, and enzyme concentration. FDH samples were incubated in the
presence of various compounds at room temperature as described under
``Experimental Procedures.'' At the indicated times, aliquots
of the solutions were taken for activity measurements. Activities
remaining are expressed as percent of initial activity. A,
samples contained 3 µg of FDH per 100 µl. Buffers were 50
mM MES/NaOH, pH 6.0, or 50 mM HEPES/NaOH, pH 7.5. B, 3 µg of FDH/100 µl in 50 mM MES/NaOH, pH
6.0 (), 3 µg of FDH/100 µl in 50 mM HEPES/NaOH, pH 7.5, 10 mM sodium formate (
); 300
µg of FDH/100 µl in 50 mM HEPES/NaOH, pH 7.5, 10
mM sodium formate (
).
To test the stability of reduced enzyme at high pH in the presence of oxygen, FDH samples were exposed to air for 2 h (Fig. 4). After this treatment, no activity was detected in the reduced FDH samples at any concentration of the enzyme studied. Interestingly, as isolated FDH was much less sensitive to oxygen. In one experiment, 94% of initial activity remained in the enzyme sample after a 1-h exposure of FDH (6 mg/ml protein) to air. Concentrated FDH (3 mg/ml) was more stable than enzyme diluted 100-fold (0.03 mg/ml), although even diluted enzyme had significant activity (Fig. 4).
Figure 4: Oxygen sensitivity of FDH. FDH samples of varying enzyme concentration in the presence or in the absence of sodium formate were prepared and exposed to air as described under ``Experimental Procedures.'' Activities of FDH samples are expressed as percent of activity of a FDH control sample which was not exposed to air.
The previously observed greater sensitivity of an isolated FDH toward oxygen exposure (16) could possibly be explained by the presence of oxygen-sensitive reduced FDH species in native FDH preparations. This is consistent with our observations that a low reduced Mo(V) EPR signal could be detected in certain isolated FDH preparations.
Figure 5:
Crystal of formate dehydrogenase H. The
crystal grew in approximately 1 week and measured 0.3 0.3
0.4 mm in size. The direction of a 4-fold symmetry axis is
shown with an arrow.
Crystals were first frozen in liquid nitrogen before removal from the anaerobic chamber for data collection. Once frozen, these crystals appear to be insensitive to the level of oxygen and thus can be handled outside the anaerobic chamber. A number of cryoprotectant solutions, including ethylene glycol, PEG 400, sucrose, and glycerol at different concentrations, were tested to obtain satisfactory freezing of the crystals. The most effective reagents for cryofreezing of FDH crystals contain 25%-30% glycerol or sucrose with 1.7 M ammonium sulfate, 1% PEG 400, and 0.1 M HEPES/NaOH at pH 7.5. Glycerol solution gave less mosaicity of diffraction than the sucrose solution. The requirement of a high concentration of glycerol is thought to be necessary to suppress a high spontaneous crystal-forming tendency of ammonium sulfate during cryofreezing, and the elevated concentration of ammonium sulfate might be needed to stabilize crystals in 25-30% glycerol. In order to reduce the crystallization tendency of ammonium sulfate and reduce the amount of glycerol used, we subsequently replaced ammonium sulfate in the cryoprotectant with lithium sulfate and achieved the same or better diffraction of crystals using 1.4 M lithium sulfate and 20-25% glycerol(25) . Both a step transfer procedure, in which a crystal was transferred stepwise to 5, 10, 15, 20, and 25% glycerol solutions, and a quick exchange procedure, in which a crystal was directly transferred into the final cryopreserving solution, were tested for applying the cryopreserving solution. Only the quick exchange procedure gave satisfactory cryofreezing results.
Figure 6:
A 1-degree oscillation image of a native
FDH crystal. The image was taken with an R-AXIS detector at a crystal
to detector distance of 175 mm and a 5-degree 2 swing angle. The
exposure time was 30 min.
The redox states of cofactors in FDH crystals were studied with EPR spectroscopy. The signal of a single reduced FeS cluster was observed at 35 K with components corresponding to those of the FeS cluster of FDH in solution (Fig. 7). The FeS signal in the crystal was dependent on the orientation of the sample in the magnetic field of the EPR spectrometer. The complexity of the FeS cluster signal of crystallized FDH suggests that FeS clusters of several orientations are present in the crystal. None of the EPR signals presented in Fig. 7, spectra b and c, were observed at 130 K, providing further evidence that these signals are derived from FeS clusters. The Mo(V) signal which is observed in solutions of FDH both at 35 and 130 K (Fig. 7, spectrum a) was not detected in crystals at these temperatures; therefore, FDH crystals do not contain Mo(V) species. These data suggest that the Mo(IV)- and reduced FeS cluster-containing form of the enzyme was crystallized.
Figure 7:
EPR spectra of FDH in solution and FDH
crystals. a, EPR signal of the frozen solution of FDH. Enzyme
was treated with 10 mM formate for 1 min. The spectrum is a
superposition of FeS
EPR signal with g-factors of 1.840, 1.957, and 2.04, and Mo(V) EPR signal with g-factors of 2.094 and 2.0. The first signal can be detected
at temperatures below 50 K, whereas the second signal can be observed
at 130 K and below. b, EPR spectrum of a single frozen FDH
crystal obtained as described under ``Experimental
Procedures.'' c, same as b but the sample was
turned by 45 degrees in the magnetic field of the EPR spectrometer.
Before plotting, spectrum c was multiplied by 0.25. Spectra a, b, and c were recorded at 35 K at
microwave powers of 0.81, 3.8, and 3.8 milliwatts,
respectively.
Although sodium formate was essential for FDH crystallization, the crystal remained intact when equilibrated in a similar solution lacking sodium formate. When a formate-free crystal was oxidized by 0.1-10 mM benzyl viologen, the crystal developed a dark blue color due to the reduced benzyl viologen. It was observed that reduced benzyl viologen binds tightly to FDH crystals and could not be easily oxidized with aerobic solutions. EPR spectroscopy was used to follow oxidation of crystals. A signal of reduced FeS cluster was observable after equilibration of the crystals in formate-free solutions. However, neither a Mo(V) signal nor a reduced FeS cluster signal was detected after a 30-min incubation of formate-free crystal in 10 mM benzyl viologen.
Ammonium bicarbonate (100 mM) was also tested as an oxidant for formate-free reduced crystals. Although the reduction of FDH by formate in solutions is reversible(17) , added bicarbonate did not reproduceably cause oxidation of crystals, presumably due to the low concentration of carbon dioxide in equilibrium with bicarbonate at pH 7.5. Oxidation of crystals from the Mo(IV)-reduced FeS cluster form to the Mo(VI)-oxidized FeS cluster form of the enzyme or incubations of oxidized crystals with inhibitors (azide, nitrite, and hypophosphate) did not change the diffraction quality of crystals. Since crystals of oxidized FDH could be obtained from formate-reduced crystals, a potential to solve the structures of both reduced and oxidized FDH is provided.
The studies described here for development of an improved purification and stabilization protocol have resulted in the first crystallization of a substrate-reduced molybdenum- and selenium-containing enzyme in its active form.