From E. C. Slater Institute, Biochemistry,
University of Amsterdam, Plantage Muidergracht 12, NL-1018 TV Amsterdam, The Netherlands and the ¶ Department of
Chemistry, State University College of New York,
Buffalo, New York 14222
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ABSTRACT |
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Infrared-spectroscopic studies on the
[NiFe]-hydrogenase of Chromatium vinosum-enriched in
15N or 13C, as well as chemical analyses, show
that this enzyme contains three non-exchangeable, intrinsic, diatomic
molecules as ligands to the active site, one carbon monoxide molecule
and two cyanide groups. The results form an explanation for the three
non-protein ligands to iron detected in the crystal structure of the
Desulfovibrio gigas hydrogenase (Volbeda, A., Garcin, E.,
Piras, C., De Lacey, A. I., Fernandez, V. M., Hatchikian,
E. C., Frey, M., and Fontecilla-Camps, J. C. (1996)
J. Am. Chem. Soc. 118, 12989-12996) and for the low spin character of the lone ferrous iron ion observed with
Mössbauer spectroscopy (Surerus, K. K., Chen, M., Van der
Zwaan, W., Rusnak, F. M., Kolk, M., Duin, E. C., Albracht,
S. P. J., and Münck, E. (1994) Biochemistry
33, 4980-4993). The results do not support the notion, based upon
studies of Desulfovibrio vulgaris [NiFe]-hydrogenase (Higuchi, Y., Yagi, T., and Noritake, Y. (1997) Structure
5, 1671-1680), that SO is a ligand to the active site. The occurrence
of both cyanide and carbon monoxide as intrinsic constituents of a
prosthetic group is unprecedented in biology.
Hydrogenases catalyze the reversible splitting of dihydrogen
(H2 With regard to the overall metal content three classes of hydrogenases
can be discriminated. The majority of hydrogenases contain nickel in
addition to iron and are termed [NiFe]-hydrogenases. The minimal
protein unit required for activity contains two subunits, a large one
(46-72 kDa) and a small one (23-38 kDa; for review see Ref. 1). The
three-dimensional structure of the enzyme from Desulfovibrio
gigas disclosed (2, 3) that the active site is a Ni-Fe dinuclear
center attached to the large subunit via four thiolates from Cys
residues. The iron atom has three non-protein ligands, with an electron
density equivalent to diatomic molecules. The small subunit contains
two [4Fe-4S] clusters and one [3Fe-4S] cluster. From a comparison
of the amino acid sequences of [NiFe]-hydrogenases, it can be
concluded that only the cubane cluster closest to the active site is
conserved in all enzymes (1).
FTIR1 studies (4-6) showed
that [NiFe]-hydrogenases contain a set of three infrared absorption
bands in the 2100 to 1850 cm A second class forms the [Fe]-hydrogenases (for review see Ref. 8);
no other metal than iron is present in these enzymes. The prosthetic
groups are located in only one subunit and minimally consist of two
classical [4Fe-4S] clusters and a hydrogen-activating site, called
the H cluster. The latter active site was speculated to be an Fe-S
cluster with 4-7 iron atoms (9, 10). It was recently discovered (6)
that also [Fe]-hydrogenases show FTIR bands in the 2100 to 1850 cm The third class of hydrogenases does not contain any metal and occurs
in methanogenic Archaea (11, 12). These enzymes, H2-forming
N5,N10-methylenetetrahydromethanopterine
dehydrogenases, can activate H2 only in the presence of
their second substrate.
In this paper we present spectroscopic as well as chemical evidence
that the molecules observed in the FTIR spectra of
[NiFe]-hydrogenases are one CO and two CN Enzyme Preparation
C. vinosum (strain DSM 185) was grown in a 700-liter
batch culture as described (14). For 15N or 13C
enrichment, cells were grown in 10-liter batch cultures with 20 mM 15NH4Cl as nitrogen source or 58 mM NaH13CO3 as carbon source.
Isotopes were purchased from Cambridge Isotope Laboratories (Cambridge,
UK). Three different cultures were prepared with final, calculated
enrichments of 98% 15N, 49% 15N, or 99%
13C. The cultures were maintained at pH 7.5 by the addition
of 1 M sodium phosphate buffer. Cells were harvested, and
the enzyme was isolated and purified as described previously (14). Due to the limited amounts of isotope-enriched samples, it was decided to
omit the TSK-DEAE column step in the purification procedure. The
Ultragel ACA-44 column was replaced by a Hiload 16/60 Superdex 200 one.
The purity of these samples, as determined by SDS-polyacrylamide gel
electrophoresis (12%) (15), was therefore only 40-60%. Enzyme was
dissolved in 50 mM Tris·HCl (pH 8.0) and stored in liquid nitrogen. Protein concentrations were determined according to Ref. 16,
using bovine serum albumin (BSA) as standard.
EPR Measurements
X-band EPR measurements (9 GHz) were obtained with a Bruker ECS
106 EPR spectrometer, equipped with an Oxford Instruments ESR-900
helium-flow cryostat with an ITC-4 temperature controller. The magnetic
field was calibrated with an AEG Magnetic Field Meter. The frequency
was measured with an HP 5350B Microwave Frequency Counter.
FTIR Measurements
FTIR measurements were performed on a Bio-Rad FTS 60A
spectrometer equipped with an MCT detector. The spectra were recorded at room temperature with a resolution of 2 cm Measurement of Hydrogenase Activity
Hydrogenase activities were determined as previously (14). The
C. vinosum enzyme samples used in this study displayed
specific H2 uptake activities (after activation under
H2 for 30 min at 45 °C) with benzyl viologen as acceptor
in the range of 100-200 µmol of H2/min/mg (30 °C, pH
8.0).
Metal Content Determination
Iron and nickel were determined with a Hitachi 180-80 polarized
Zeeman Atomic Absorption Spectrophotometer using a standard series of
the particular metal. Adventitious metal ions were eliminated from
enzyme and buffer by passage through a Chelex 100 column (Bio-Rad).
Relative metal contents were found to be 10-13 iron per nickel, in
line with the presence of one iron atom in the active site and two
[4Fe-4S] and one [3Fe-4S] clusters. The protein concentrations
determined by the Bradford method correlated well with the values based
on the metal contents, using a molecular mass of 94 kDa (14).
Sample Preparation
Usually, hydrogenase of C. vinosum as isolated in air
is a mixture of two forms, called ready and unready, with quite
different EPR spectra and slightly different FTIR spectra. Therefore,
enzyme was converted to more than 90% into the ready form as follows. A dilute solution (10 µM) was incubated under 1.2 bar of
H2 at 50 °C for at least 30 min, cooled to 2 °C,
evacuated, and flushed twice with argon, after which argon was
substituted by 1.2 bar of O2. After stirring for 10 min at
2 °C and 60 min at room temperature, the sample was concentrated by
means of a Microcon PM10 to about 300 µl and inspected by EPR to
verify the conversion into the ready form. The sample was recovered
from the EPR tube and concentrated to 10 µl. As some spin coupling of
nickel with the {Xox = [3Fe-4S]+} moiety (X is an unknown,
n = 1 redox component which, when oxidized, is strongly
spin coupled to the oxidized [3Fe-4S]+ cluster (7)) was
detected, it was treated with freshly prepared phenazine methosulfate
(5 µM) and neutralized ascorbic acid (25 mM),
which removes this coupling by reduction of the unknown group X. The sample was loaded into the IR transmittance cell and
incubated for 30 min at room temperature before recording the spectrum. No reduction of the active site itself was noticed in the FTIR spectra
(5).
Before use, H2 was passed over a Palladium catalyst
(Degussa, Hanau, Germany; type E236P), and argon was passed through an Oxisorb cartridge (Messer-Griesheim, Düsseldorf, Germany) to remove residual O2.
Determination of Protein-bound CO
The direct demonstration of CO(g), released from
hydrogenase upon denaturation was performed by binding to ferrous
hemoglobin, essentially as described earlier (17). Typically,
hydrogenase of C. vinosum (2.5-5 nmol) was denatured by
treatment with 5% SDS under an argon atmosphere in 700-µl vials with
lined aluminum seals. The sample was incubated for 10 min at 95 °C.
After cooling, the vial was incubated for 60 min at room temperature
under continuous stirring. After a short centrifugation to spin down
any water droplets, the total gas phase in the head space was carefully withdrawn with a gas-tight Hamilton syringe, while at the same time
water was injected with another syringe to balance the under-pressure. The removed gas was injected into an anaerobic cuvette, filled with an
assay mixture containing 0.9 mg/ml hemoglobin, 100 mM NaCl,
8 mM sodium dithionite, and 25 mM CAPSO buffer
(pH 9.5) under an argon atmosphere (17). The assay mixture was
equilibrated with the gas phase for 30 min at room temperature, by
repeatedly turning the cuvette upside down on a rotating tilted plate.
Then the absorption spectrum was recorded from 400 to 460 nm on a
Hewlett-Packard 8452A Diode Array Spectrophotometer, using a cuvette
without added CO as background. Binding of CO to reduced hemoglobin
leads to a blue shift of the Soret band (increase in the extinction
coefficient for Hb at 420 nm from 109.5 to 192 mM Determination of Protein-bound CN A common approach to determine cyanide in aqueous solutions is
to acidify the sample with sulfuric acid, followed by heating of the
mixture. The evolved HCN gas (boiling point, 299 K) is transported by
means of a carrier gas and led through an alkaline solution, which
absorbs the HCN (18). The collected cyanide can be determined by
spectrophotometric procedures based on a modified König's
reaction (19), which starts with the production of cyanogen chloride.
This is followed by a reaction of the cyanogen chloride with a pyridine
derivative resulting in ring opening, thereby producing a 2-pentenedial
derivative. Subsequently, a condensation reaction between the
2-pentenedial derivative and an active methylenecarbonyl compound is
performed, e.g. with barbituric acid, with pyrazolone or
with 2-thiobarbituric acid (19). In the present work the isonicotinic
acid/barbituric acid method was applied (Fig. 1
), modified from Nagashima (19), using
1,3-dimethylbarbituric acid as described (20), under less extreme
conditions (18). The blue product of the latter reaction is
polymethine, having an absorption maximum at 600 nm. Color development
using 1,3-dimethylbarbituric acid is faster and more intense compared
with that of barbituric acid (20).
INTRODUCTION
Top
Abstract
Introduction
References
2H+ + 2e
) and are common
in many microorganisms. Their physiological role is either to acquire
reducing equivalents from H2 or to dispose of excess
reducing equivalents from fermentation via the reduction of protons.
Hydrogenases are often intimately complexed to modules containing other
redox proteins. In this way the metabolism of dihydrogen is linked to
redox chemistry with a wide variety of electron acceptors and donors
like NAD(P)(H), b- and c-type cytochromes, factor
F420, S2
, and membrane-bound (mena)quinones.
1 spectral region, not found
in any other proteins. As the frequency of these bands is very
sensitive to the status of the active site, it was concluded that they
are due to intrinsic ligands (diatomic molecules with a triple bond or
triatomic molecules with two adjacent double bonds) of the active site.
Also a unique lone low spin Fe(II) site was detected, in addition to
the high spin iron sites of the Fe-S clusters, by Mössbauer
spectroscopy (7).
1 spectral region, which strongly shift upon changes of
the redox state of the enzyme. Hence a similar architecture was
suggested for the active sites of [NiFe]- and
[Fe]-hydrogenases.
groups bound
to iron in the Ni-Fe active site. A preliminary report of this work has
appeared elsewhere (13).
EXPERIMENTAL PROCEDURES
1 and are
averages of 762 or 1524 scans. Enzyme samples (10 µl, 0.4-1.5
mM) were loaded into a gas-tight IR-transmittance cell (4)
with polished CaF2 windows kept at 50 µm distance with a
Teflon spacer. A cell containing 50 mM Tris·HCl (pH 8.0)
was used as reference. The small, variable contributions of water vapor
was removed by subtraction of an appropriate water-vapor spectrum. The
multi-point method of the Bio-Rad spectrometer was used for correction
of the base line.
1·cm
1 and decrease of the
extinction coefficient for Hb at 430 nm from 140 to 60 mM
1·cm
1 (17), and hence a
E(420-430 nm) = 162.5 mM
1·cm
1 was used for
quantification of the formed Hb-CO species. The recovery of the overall
procedure tested with samples of CO-saturated water was 91%.
View larger version (16K):
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Fig. 1.
Summary of the reactions involved in the
spectrophotometric assay for the determination of cyanide in
hydrogenase.
The determination of cyanide in the hydrogenase of C. vinosum was performed by mild oxidative treatment with potassium permanganate in sulfuric acid (18), followed by detection of cyanide as described above. The methods have been optimized for the use with iron-sulfur proteins as described below.
Release of Cyanide from the Enzyme by Treatment in a Mildly Acidic and Oxidizing Environment-- An appropriate setup was built using a 10-ml double-necked round-bottom flask, filled with 4.3 ml of distilled water, 20 µl of 3% (w/w) silicone anti-foaming agent (BDH, Poole, UK), and 250 µl of 3 M H2SO4. A small magnetic stirring bar was used for mixing. The flask contained a gas inlet, consisting of a thin glass tube reaching to the bottom. During the reaction, a flow of argon gas was bubbled through the solution (20-30 ml/min).
As acid-labile sulfur is released upon destruction of iron-sulfur
clusters in the protein by acid, it is likely, and was actually observed, that any S0 formed reacts with cyanide to form
thiocyanate (21). This would reduce the yield of the cyanide from the
enzyme. We therefore added up to 200 µl of 5 mM
KMnO4 prior to heating in order to re-oxidize possible
SCN to CN
in the sulfuric acid environment
(18). The reaction mixture was heated in a water bath to 95 °C,
after which 50-250 µl of hydrogenase (about 10 nmol) was added by
injection through a septum. Heating under a steady flow of argon was
continued for 30 min. The carrier gas was led through 1 ml of 100 mM NaOH to collect the HCN. The tube was weighed before and
after the experiment to correct for evaporation.
Oxidation of Cyanide with Chloramine T to Form Cyanogen Chloride-- In a reaction tube, 0.5 ml of the NaOH solution containing the collected HCN, 1.5 ml of distilled water, 0.5 ml of succinate solution (2 M), and 0.05 ml of 1% chloramine T were mixed and kept for about 1 min at room temperature at pH 5.6. Because of the volatility of cyanogen chloride, it was desirable to add the color reagent as soon as its formation had reached a maximum, which was after about 1 min, as also found previously (22). The succinate solution (2 M succinic acid, 2.6 M NaOH) was prepared by slow addition of NaOH to a cold suspension of succinic acid. Afterward the solution was filtered.
Reaction of Cyanogen Chloride with Isonicotinic Acid and 1,3-Dimethylbarbituric Acid to Form Polymethine-- A color reagent solution was prepared by adding 2.8 g of 1,3-dimethylbarbituric acid and 2.3 g of isonicotinic acid to 150 ml of distilled water. Solid NaOH (~1.3 g), required to dissolve the acids (23) and to adjust the pH to 5.6, was then slowly added, thereafter the solution was diluted to 250 ml with distilled water. To the mixture as obtained under "Oxidation of Cyanide with Chloramine T to Form Cyanogen Chloride," 0.45 ml of color reagent was added and left for 30 min at room temperature. The reaction mixture was then transferred to a glass cuvette, and the polymethine concentration was determined by measuring its absorbance at 600 nm using a Zeiss M4 QIII spectrophotometer. A sample of 0.5 ml of pure 100 mM NaOH, treated in the same way, was used as a blank. A series of KSCN samples (0-20 nmol), prepared from a 0.0998 M solution in water (volumetric KSCN standard, Aldrich), was used as a standard curve. This curve was linear in the used range and gave precisely the same color as a series of KCN solutions but is more convenient for routine analyses.
It was observed that beyond a certain concentration of KMnO4, the amount of HCN released from hydrogenase increased steadily (Fig. 2 ). By using BSA, it was found that HCN could develop from pure protein as well, if the concentration of KMnO4 used per mg of protein exceeded a certain value (about 0.5 mM per mg; Fig. 2). Therefore, routinely cyanide determinations were carried out using 0.05-0.2 mM KMnO4 and 0.5-1 mg of protein. With these amounts virtually no HCN was released from protein (Fig. 2). The recovery of KCN or KSCN added to the distillation setup as described above was determined to be at least 90%; therefore, no correction for loss during distillation of the cyanide was made.
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Exchange Experiments with 13CN
In order to test whether the IR-detectable groups could be
exchanged with added cyanide, hydrogenase of C. vinosum was treated with K13CN (Aldrich). One sample
was incubated with 5 mM K13CN for 24 h at
30 °C in air at pH 8.0, and directly loaded to the IR transmittance
cell. A similar incubation was carried out in the presence of 8 M urea, in order to loosen the protein structure to a
certain extent (C. vinosum hydrogenase is stable in 8 M urea (24)) and to possibly make the active site more
accessible. Prior to the measurements urea was removed from the sample
by dialysis. In another experiment an incubation with 2 mM
K13CN was carried out with enzyme activated under
H2 for 30 min at 50 °C.
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RESULTS |
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FTIR Spectra of Hydrogenase Enriched in 15N or
13C--
In Fig. 3 , trace A, the FTIR spectrum of C. vinosum
[NiFe]-hydrogenase in the ready form is shown. Two small bands (2090 and 2079 cm1) and one large band (1944 cm
1)
can be seen. Enzyme enriched in 13C showed the spectrum in
trace B. The large band was shifted to 1899 cm
1, whereas a small band (1943 cm
1) was
still detectable at the original position. The two bands at 2090 and
2079 cm
1 shifted to 2046 and 2035 cm
1. It
is concluded that all groups responsible for the IR bands in
trace A contain carbon.
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Enrichment of the enzyme with 15N resulted in a shift of
the two small bands (Fig. 3, trace C) to 2060 and 2049 cm1. The position of the large band was not affected.
Hence, the groups responsible for the two small IR absorption bands
contain nitrogen, whereas the group evoking the large absorption band does not. All band positions are summarized in Table
I.
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FTIR Spectra of Hydrogenase Enriched 49% in
15N--
The results in Fig. 3 indicate that the two small
bands are presumably due to CN groups. The two bands may
reflect the symmetrical and antisymmetrical vibrations of two
vibrationally coupled CN
groups (25, 26) or they might be
due to two different conformers of the enzyme, each with only one
CN
ligand in the active site. Recently, a high resolution
crystal structure (1.8 Å) has been published of the
[NiFe]-hydrogenase from Desulfovibrio vulgaris, Miyazaki
(27). Although the overall structure of the enzyme and its active site
highly resembled the structure of the D. gigas enzyme (3),
it was concluded that the diatomic ligands at the iron atom were one
CO, one CN
, and one SO group. FTIR spectra of this enzyme
were very similar to those observed with the C. vinosum and
D. gigas enzymes, and this was explained by assuming that
the two bands in the 2100-2050 cm
1 region were due to
two different conformers of the enzyme (27). To verify this possibility
in the C. vinosum enzyme, we have also prepared enzyme
enriched 49% in 15N. The result is shown in Fig. 4
, trace B. For a proper
understanding, the spectra of unenriched enzyme (trace A)
and fully 15N-enriched enzyme (trace C) are
shown in the same figure as well. In case of two conformers of enzyme
with one CN
ligand only, a mixture of the spectra in
A and C would be expected. This is clearly not
the case. Instead, trace B shows two sets of three
overlapping bands in the 2100 to 2040 cm
1 region. Two of
the three bands in each set are at positions equivalent to those seen
in traces A or C. The central band in each set
can be visualized by appropriate subtractions (Fig. 4, trace
D). The two bands, at 2085 and 2053 cm
1, are
somewhat broader than those of the individual bands of the unenriched
or fully enriched enzyme.
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EPR-- Possible effects of isotope enrichments on the EPR spectra of the nickel center and the [3Fe-4S]+ cluster in the ready or unready form of the C. vinosum enzyme as isolated have been studied as well. The preparations enriched for more than 98% in 13C or 15N did not show any detectable broadening as compared with unenriched enzyme, however.
Chemical Analysis for CO and CN--
To corroborate the
implications of FTIR experiments, analytical procedures for the
determination of intrinsic CO and CN in proteins and in
particular hydrogenase have been designed. We encountered several
complications. First, release of CO from the C. vinosum
[NiFe]-hydrogenase was not observed using a variety of denaturing,
aerobic, acid, alkaline, and temperature conditions other than the
current anaerobic, neutral pH, SDS (100 °C) procedure. Although the
method itself has a high recovery (91%), only up to 0.7 mol of carbon
monoxide per mol of nickel was detected (Table II). When Hb-CO samples were used as a
control, lower amounts of CO were also found (0.4-0.8 mol/mol Hb). No
carbon monoxide evolution was seen in control experiments with other
proteins. Second, the release of CN
from hydrogenase was
initially corrupted by reaction of acid-released cyanide with sulfur to
form non-distillable thiocyanate. The subsequent use of
KMnO4 to oxidize SCN
back to CN
led to side reactions with amino acids like tryptophan, tyrosine, cystine, and cysteine (18). The release of cyanide as a function of the
quantities of permanganate, hydrogenase, and the non-cyanide-containing model protein BSA was therefore investigated (Fig. 2). By the use of
0.5-1.0 mg of protein amounts and low (0.05-0.2 mM)
KMnO4 concentration-sensitive and specific detection of the
cyanide contained in the hydrogenase active site could be achieved. The characteristics of the aspecific generation of cyanide from BSA and
hydrogenase above 0.5 mM KMnO4 are so similar
that it can be assumed that the cyanide released in the 0-0.2
mM KMnO4 range corresponds to cyanide not
generated from amino acid breakdown but from SCN
derived
from reaction of CN
with sulfur compounds. The above is
exemplified by the very reproducible nature (six preparations) and
limiting stoichiometry of 2 mol of cyanide per mol of nickel (Table
II), mutually consistent with the FTIR results and x-ray
crystallography.
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Non-exchangeability of CO and CN--
No changes in the IR spectra
of C. vinosum hydrogenase were observed upon incubation with
5 mM K13CN under the conditions tested
(i.e. incubation of oxidized hydrogenase at 30 °C with 5 mM K13CN in air for 24 h, in the presence
or absence of 8 M urea, or incubation of
H2-activated enzyme with K13CN). In our
previous studies (4) it was already found that incubation of inactive,
oxidized enzyme or active, reduced enzyme with 13CO did not
shift or replace any of the FTIR bands either. This means that the
CN groups and the CO molecule are not exchangeable under
the examined conditions.
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DISCUSSION |
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FTIR Spectra--
The ready and unready forms of C. vinosum hydrogenase both have FTIR spectra with three bands in the
2150 to 1900 cm1 region, be it with slightly different
positions (2090, 2079, and 1944 cm
1 for the ready enzyme
and 2093, 2083, and 1945 cm
1 for the unready form). The
previous preliminary results (13) were performed with mixtures of ready
and unready enzyme. For a proper comparison of the effects of isotopes,
we have therefore converted the enzyme samples into the ready form and
so the current data have an increased accuracy for the isotope shifts.
By using the equation for a classical harmonic oscillator,
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(Eq. 1) |
As previously stated, partial labeling of the center can be used to
distinguish whether the two CN bands detected arise from
two CN
ligands on a single metal center or whether they
arise from two different conformers of a single CN
ligand. The rationale is as follows. In the case of two
CN
ligands located on the same active site iron,
vibrational coupling of the CN
vibrations is expected.
Partial labeling of the CN
ligands using 15N
(giving rise to a population of molecules containing one
C14N ligand and one C15N ligand) will result in
the partial loss of this coupling. This is in turn expected to give
rise to detection of additional bands in the partially labeled spectra
at frequencies intermediate to the bands arising from the fully
unlabeled (C14N/C14N) and fully labeled
(C15N/C15N) cases. Hence for a spectrum of a
center with a partial C15N label, assuming that the
CN
ligands are in identical environments, a total of 6 infrared bands attributable to CN
should be detected. Two
of these bands are attributable to the case where both CN
ligands contain 14N (C14N/C14N) and
will result in frequencies identical to those detected in the
unenriched enzyme. Two of these bands will be due to centers in which
both CNs contain 15N (C15N/C15N)
and will have frequencies identical to the bands detected in the fully
labeled enzyme. The remaining two bands will arise from enzyme
containing one CN
labeled with 14N and one
CN
labeled with 15N
(C14N/C15N). These bands will be located at
frequencies slightly higher than the average of the frequencies for the
bands arising from the C14N/C14N case and
slightly lower than the average frequency of the bands arising from the
C15N/C15N case. In comparison, if the two
CN
bands detected arise from two conformers of a single
CN
ligand on the active site iron, then the pattern of
bands detected in a partial labeling experiment will consist of a total
of 4 infrared bands, having frequencies arising only from two
C14N and two C15N. Hence, the resultant spectra
should look like a sum of the infrared spectra for the unlabeled and
fully labeled enzyme. Enrichment of the enzyme with 49%
15N (Fig. 4) clearly shows that the two small bands are not
due to two different conformers of the enzyme with only one cyanide but
that they arise from two vibrationally coupled cyanide groups bound to
the same metal ion. The resultant spectra for partially labeled enzyme
consists of a total of 6 bands. The relative intensity of the three
bands in each of the two sets in the 2100 to 2040 cm
1
region is as expected from a mixture of enzyme molecules with 26%
C14N/C14N, 50%
C14N/C15N, and 24%
C15N/C15N (49% 15N enrichment).
The bands of enzyme molecules with a C14N/C15N
couple are at 2085 cm
1 (C14N) and 2053 cm
1 (C15N) (Fig. 4, traces B and
D). The frequencies are in good agreement with what is
expected for partially labeling a center containing two coupled
CN
ligands, a small residual coupling being noticed even
after this labeling. The resultant bands are, however, somewhat broader
than those in the traces A and C. This is
presumably caused by slight differences in the protein environment of
the two CN
ligands, as apparent from the x-ray structure,
i.e. a CN hydrogen-bonded to a Ser residue and a CN
hydrogen-bonded to an Arg residue (3, 28).
From the data in Figs. 3 and 4 we therefore conclude that the two small bands arise from two coupled cyanides and the large one from CO.
Chemical Analysis of CO and CN--
The procedure to
detect enzyme-bound CO is relatively straightforward. About 0.7 mol of
CO/mol of nickel was found in C. vinosum [NiFe]-hydrogenase. The recovery of the method is quite good (91%), so it is unclear why less than 1 CO per nickel was found. It cannot be
excluded that part of the CO is oxidized to CO2 during
denaturation of the enzyme, when the prosthetic groups degrade to a
uncontrolled mixture of iron, nickel, sulfur, and CN
.
Lower amounts were also obtained with Hb-CO as a control. The chemical
analysis quite clearly showed the presence of 2.0 mol of
CN
/mol of nickel.
Structure of the Active Site--
From the combined results of
present and previous (4-6) infrared spectroscopic and chemical
analyses, it is concluded that the active site of C. vinosum
[NiFe]-hydrogenase contains two cyanides and one carbon monoxide
bound to the same metal ion. As most other [NiFe]-hydrogenases have
basically the same infrared spectrum (6, 29), this conclusion can be
extended to those enzymes as well. The x-ray structure of the D. gigas enzyme (2, 3) shows that these diatomic ligands are bound to
iron in the bimetallic NiFe site. Our data do not support the
suggestion of Higuchi et al. (27), based on studies of the
D. vulgaris enzyme, that [NiFe]-hydrogenases have only one
CN as a ligand to iron, in addition to SO and CO.
The results of our study enable a refinement of the model of the active site of [NiFe]-hydrogenases provided by the crystallographic studies (2, 3). This is shown in Fig. 5 . The strong ligand field due to the ligands around the active-site iron is expected to render it low spin, and this explains and confirms the observation of a lone low spin ferrous iron by Mössbauer spectroscopy (7) in the C. vinosum enzyme. This is unlike the remaining iron atoms in the enzyme, which are all contained in Fe-S clusters, and hence are high spin.
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In view of the presence of similar infrared bands in [Fe]-hydrogenases (6), CO and cyanide are expected to be part of the active site in that class of hydrogenases as well. Hence, the activation of molecular hydrogen by all presently known, metal-containing hydrogenases obviously requires cyanide and carbon monoxide as ligands to iron in the active sites. The precise nature of these ligands in [Fe]-hydrogenases, as well as their stoichiometry, are currently under investigation in our laboratory.
Metal Cyanide Versus Metal Isocyanide--
By examining the
crystal structure, Volbeda and co-workers (3, 28) noted that ligand L1
could accept H bonds from the OH group of Ser-486 and its peptide NH
and that L2 might form H bridges to the guanidine moiety of Arg-463, as
well as to its peptide NH. Arg-463 is strictly conserved in
[NiFe]-hydrogenases (30). At the position where serine is found in
the D. gigas enzyme, either serine or threonine, which
contains an hydroxyl group as well, is found in other
[NiFe]-hydrogenases. Ligand L3 only makes contact with hydrophobic
residues (3, 28), and this is why CN is preferred at the
positions of L1 and L2. If the cyanide groups are indeed H-bridged,
then these ligands will coordinate as cyanides, Fe-C-N···H, and
not as isocyanides.
In inorganic compounds, iron very rarely complexes CN as
isocyanide, due to the instability of the formed complex. The highest occupied sigma orbital is localized on the carbon atom, making this
atom more basic than the nitrogen atom. This makes the M-C configuration the most stable one (31). In iron-containing
organometallic complexes CN
is able to bind as isocyanide
and almost exclusively if the cyanide bridges between two metals, using
both its carbon and nitrogen for binding. The crystal structures of the
D. gigas (3) and D. vulgaris (27) hydrogenases
rule out such a possibility.
Non-exchangeability of CO and CN--
The
crystallographic studies (3, 28) revealed that the diatomic ligands are
tightly buried in narrow protein cavities, the size of which was
estimated to be insufficient to hold triatomic molecules. This is
probably the reason why we could not detect any exchange of the
diatomic molecules with either 13CO or
13CN
and why the groups are so tightly
associated with the enzyme.
Localization of the Unpaired Spin in the Active Site--
From the
observation that no detectable broadening of the nickel EPR signal
could be observed in either 13C- or
15N-enriched enzyme, it is concluded that the unpaired spin
in the active site has no appreciable spin density on carbon or
nitrogen atoms from amino acid residues or the CN/CO
ligands. As also no broadening is observed in samples enriched in
57Fe, this underlines the conclusion (1) that the unpaired
spin is localized on the nickel ion and its immediate ligands in the active site.
Model Compounds--
Recently two interesting iron model complexes
with CN and CO ligands have been described as a reaction
in our preliminary report (13). One is a low spin Fe(II) thiolate
complex with one CN
and one CO as ligands (32), this
being the first example of this kind. The CN
and CO
stretch frequencies were found at 2079 cm
1 and 1904 cm
1, respectively. A second, even more interesting model
is an iron compound in which iron is sandwiched between a
cyclopentadiene ring and two cyanides and one carbon monoxide (33). The
potassium salt of this compound matches the structural and infrared
characteristics of the Fe(CN)2CO site in
[NiFe]-hydrogenases (33), having bands at 2094, 2088, and 1949 cm
1 in aprotic media. As in the enzyme, very little
vibrational coupling was observed between the v(CN) and
v(CO) modes in this complex. The bands in the model compound
are clearly broader than those observed in the enzymes.
Biosynthesis--
Enrichment to more than 98% in either
13C or 15N resulted in a complete shift of the
v(CN) bands (Fig. 3). In the 13C-enriched
enzyme, however, about one-fifth (22%) of the v(CO) band
was still detectable at the original position. In principle, this could
form an indication that all carbon for CN formation might
come from bicarbonate, but this is not so for formation of CO. After
considering the amounts of 12C entering the culture from
other possible sources, e.g. EDTA, cells, and products from
the inoculum, we estimated these contaminations to be a few percent at
most. We therefore have no explanation for the observed effect.
The biosynthesis of [NiFe]-hydrogenases is a highly complicated
process (34, 35). A set of accessory genes in the hydrogenase operon is
known to encode for proteins involved in the maturation of this class
of enzymes. In view of the extraordinary nature of the active site
structure, it has been proposed that one or more of these gene products
are involved in capture (nickel and iron), synthesis (CO and
CN), and incorporation of the several constituents of the
active site. Indeed, Rey et al. (36) reported that one of
the accessory genes (hypX) in Rhizobium
leguminosarum codes for a protein resembling N10-formyltetrahydrofolate-dependent
enzymes involved in C1-metabolism. This indicates that the diatomic
ligands may be formed by the products of these accessory genes.
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FOOTNOTES |
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* This work was supported by the Netherlands Foundation for Chemical Research (SON) and the Netherlands Organization for Scientific Research (NWO).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: TNO Nutrition and Food Research Institute, P. O. Box 360, NL-3700 AJ Zeist, The Netherlands.
Supported by a Cotrell College Science Award of Research Corp.
** To whom correspondence should be addressed. Tel.: 31 20 525 5130; Fax: 31 20 525 5124.
The abbreviations used are: FTIR, Fourier transform infrared spectroscopy; EPR, electron paramagnetic resonance; BSA, bovine serum albumin; CAPSO, 3-[cyclohexylamino]-2-hydroxy-1-propanesulfonic acid.
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