J. Am. Chem. Soc. 1999, 121, 9457-9458 Detection of a New Radical and FeMo-Cofactor EPR Signal during Acetylene Reduction by the r-H195Q Mutant of Nitrogenase
Morten Sørlie,† Jason Christiansen,‡ Dennis R. Dean,*,‡ andBrian J. Hales*,†
Department of Chemistry, Louisiana State UniVersityDepartment of Biochemistry,Virginia Polytechnic Institute andState UniVersity, Blacksburg, Virginia 24061ReVised Manuscript ReceiVed August 19, 1999
The Mo-dependent nitrogenase of Azotobacter Vinelandii is a
two-component system consisting of iron (Fe) protein andmolybdenum-iron (MoFe) protein. In addition to the physiologi-cally relevant conversion of N2 to NH3, nitrogenase catalyzes theATP-dependent reduction of simple, multiple bonded moleculessuch as C2H2, HCN, and HN3. Substrate reduction is believed to
Figure 1. The g-2 region of the EPR spectrum of enzymatic turnover
7S9 homocitrate metal cluster called FeMo-
for R-H195Q in the presence of acetylene. Inflections originating from
cofactor which is contained within the R-subunit of the MoFe
the g ) 2.12 signal (g ) 2.12, 1.98, 1.95) are marked (a), the g ) 2.00
protein. During catalysis the Fe protein serves as a specific,
signal is marked (b), and the g ) 1.97 signal is marked (c) The whole
MgATP-dependent reductant of the MoFe protein. In its as-
spectrum is shown as the insert. Note that the S ) 3/2 FeMo-cofactor
isolated form the MoFe protein displays a rhombic S ) 3/2 EPR
signal is almost completely replaced by the S ) 1/2 signals. To obtain
signal (g ) 4.3, 3.6, and 2.0) originating at the FeMo-cofactor.
the high resolution observed in both spectra the modulation amplitude
During turnover this signal is diminished by up to 90% to an
was 0.1 mT, well below the value normally used to record spectra of
EPR-silent state. When the potent noncompetitive inhibitor CO
metal clusters. Experimental conditions: [Fe protein]/[R-H195Q] ) [0.020
is present in the turnover system, two different intense S ) 1/2
mM]/[0.100 mM] ) 1:5; [C2H2] ) 0.1 atm; [ATP] ) 10 mM; [MgCl2]
are generated,1-3 lo-CO (g ) 2.09, 1.97, 1.93; P
2S2O4] ) 20 mM; 50 mM TES-KOH, pH 7.4. Spectrometer
and hi-CO (g ) 2.17, 2.06, 2.06; P
parameters: microwave frequency ) 9.45 GHz; microwave power ) 2
signals have been investigated with ENDOR spectroscopy4-7 and
mW; modulation amplitude ) 0.1 mT; temperature ) 4K.
were shown to arise from one or two molecules of CO,respectively, bound to the FeMo-cofactor. Although minor
has received much attention since it previously has been shown
substrate-induced EPR signals have been elicited from the MoFe
that, although its phenotype for reduction of most substrates
protein under turnover conditions,2,8 to date no nitrogenase
resembles that of wild-type (acetylene has a nearly identical Km),
substrates have been shown to induce strong signals for the wild-
it has the unique property that N2 binds during turnover but is
type enzyme similar to those observed when CO is present under
not significantly reduced.9,13 This mutant MoFe protein also
turnover conditions. Herein, we describe the first report of intense
appears to be minimally altered spectroscopically because it
EPR signals, including a radical signal, that are elicited from an
exhibits a rhombic S ) 3/2 EPR signal nearly identical with that
altered form of the MoFe protein (R-H195Q) when incubated in
found for the wild-type MoFe protein in the as-isolated state. Thus,
it was worthwhile to determine if EPR signals arising from
The R-H195Q mutant form of the MoFe protein was con-
enzyme turnover events, previously unobservable in the wild-
structed, isolated, and investigated by Kim et al.9 This altered
type MoFe protein, could be observed in this altered protein.
form of the MoFe protein has glutamine substituted for the
Figure 1 shows the turnover-dependent, acetylene-induced EPR
R-subunit histidine-195 residue, which is a strictly conserved
signal of R-H195Q MoFe protein in the g-2 region (sample was
amino acid within the MoFe protein and is within hydrogen-
rapidly frozen in liquid N2 3 min following initiation of turnover)
bonding distance of the FeMo-cofactor.10-12 The altered protein
with the full spectrum included as the insert. This signal (spinintegration 0.23 ( 0.02 spins per cofactor) has inflections at g )
[2.12, 2.00, 1.98, 1.95] with a minor shoulder at g ) 1.97 and is
Virginia Polytechnic Institute and State University.
(1) Yates, M. G.; Lowe, D. J. FEBS Lett. 1976, 72, 121-126.
not detected when the wild-type enzyme is used under the same
(2) Lowe, D. J.; Eady, R. R.; Thorneley, R. N. F. Biochem. J. 1978, 173,
conditions. The numerous inflections show that this signal
originates from more than one paramagnetic species. To inves-
(3) Davis, L. C.; Henzl, M. T.; Burris, R. H.; Orme-Johnson, W. H. Biochemistry 1979, 18, 4860-4869.
tigate the relationship of these signals, turnover samples were
(4) Pollock, R. C.; Lee, H.-I.; Cameron, L. M.; DeRose, V. J.; Hales, B.
prepared in the presence of C2H2 and allowed to incubate at
J.; Orme-Johnson, W. H.; Hoffman, B. M. J. Am. Chem. Soc. 1995, 117,
different temperatures (10, 30, and 45 °C) prior to rapid freeze-
(5) Christie, P. D.; Lee, H.-I.; Cameron, L. M.; Hales, B. J.; Orme-Johnson,
quench. Figure 2 shows the EPR spectra obtained from samples
W. H.; Hoffman, B. M. J. Am. Chem. Soc. 1996, 118, 8707-8709.
incubated at 30 and 45 °C illustrating that the g ) 2.00 inflection
(6) Lee, H.-I.; Hales, B. J.; Hoffman, B. M. J. Am. Chem. Soc. 1997, 119,
has smaller amplitude relative to the g ) 2.12 inflection at 45 °C
(7) Lee, H.-I.; Cameron, L. M.; Hales, B. J.; Hoffman, B. M. J. Am. Chem.
°C. Similar changes were observed in turnover
Soc. 1997, 119, 10121-10126.
samples made at 30 °C compared to those at 10 °C (results not
(8) Rasche, M. E.; Seefeldt, L. C. Biochemistry 1997, 36, 8574-8585.
shown) indicating that the g ) 2.00 and 2.12 inflections represent
(9) Kim, C.-H.; Newton, W. E.; Dean, D. R. Biochemistry 1995, 35, 2798-
different species. Temperature-dependent and power-dependency
(10) Kim, J.; Rees, D. C. Science 1992, 257, 1677-1682. (11) Kim, J.; Rees, D. C. Nature 1992, 360, 553-560.
(13) Dilworth, M. J.; Fischer, K.; Kim, C. H.; Newton, W. E. Biochemistry
(12) Chan, M. K.; Kim, J.; Rees, D. C. Science 1993, 260, 792-794. 1998, 37, 17495-17505.
9458 J. Am. Chem. Soc., Vol. 121, No. 40, 1999
) 2.12 inflection observed in 13C2H2 turnover samples is slightlybroader than that observed for C2H2 turnover samples (SupportingInformation). These data suggest the g 2.12 signal arises from anC2H2 adduct(s) bound to the FeMo-cofactor during enzymaticturnover.
While an acetylene-induced signal has not been reported
previously, a weak axial S ) 1/2 signal (g ) [2.125, 2.000, 2.000];0.017 spins per cofactor) has been detected2 in turnover samplesof nitrogenase from Klebsiella pneumoniae in the presence ofethylene (1.0 atm; signal maximizes at 29 K and 20 mWmicrowave power). This signal is not observed in turnoversamples under identical conditions using either wild-type or
R-H195Q nitrogenase from A. Vinelandii. However, reducing thetemperature to 4 K on the R-H195Q sample reveals a weak signal(Figure 2 insert) nearly identical with that observed in the presenceof acetylene (Figure 1), strongly suggesting that the g 2.12 signalarises from ethylene bound to the FeMo-cofactor.
The possible identity of the g 2.00 signal can be further refined.
If this signal arose from an intermediate radical of reduced
Figure 2. The g-2 region of the EPR spectrum of enzymatic turnover
acetylene, the spin density would be localized on the carbon of
for R-H195Q in the presence of acetylene after incubation at t ) 30 and45 °C, respectively. Experimental and spectrometer conditions are as
the radical and a large isotope-induced change in the EPR signal
described in Figure 1. Insert: The g-2 region of the EPR spectrum of
would be expected with either 13C2H2 or C2D2. Only very small
enzymatic turnover for R-H195Q in the presence of ethylene (1.0 atm).
changes are observed (Table 1) which may originate from changes
Experimental and spectrometer conditions are as described in Figure 1.
in the underlying g 2.12 signal. In summary, the results suggestthat the g 2.12 signal arises from C2H4 bound at the FeMo-cofactor
Substrate Isotope Effects on the Line Widths of
while the g 2.00 signal is most associated with either an amino
acid or homocitrate radical species that is generated during
All three signals are observed at C2H2 concentrations as low
as 0.001 atm and the amplitude ratio of the individual inflections
remain unchanged. In other words, there are no hi-/lo-C
analogous to the aforementioned hi-CO and lo-CO CO signals.
A plot of signal amplitude vs C2H2 at low concentrations results
studies (results not shown) further demonstrate that the shoulder
in a sigmoidal curve suggesting cooperative binding of more than
at g ) 1.97 shows behavior that is drastically different from the
one C2H2. This is consistent with the works of Davis et al.3 and
other signals. These results indicate that there are at least three
Shen et al.,14 who have provided evidence for more than one C2H2
different signals present in the g-2 region. The first (termed g
binding site within the MoFe protein. Finally, similar to the
2.12) is a rhombic signal with g ) [2.12, 1.98, 1.95]. Large
formation of hi-CO under low flux conditions,15 all three
deviation from the g-factor of the free electron (g ) 2.0023) is
acetylene-induced EPR signals appear at low electron flux.
characteristic of an unpaired electron on a transition metal or metal
Increasing the component ratio resulted in an overall increase in
cluster leading to significant spin-orbit coupling. Because the
the final amplitude of the EPR signals without a major change in
substituted amino acid is located within hydrogen-bonding
the amplitude ratios of the individual inflection. Thus, the three
distance of the cofactor the g 2.12 signal most likely originates
species contributing to the spectrum in Figure 1 are simultaneously
generated in approximately the same ratio regardless of substrate
The second signal (termed g 2.00) is narrow, nearly isotropic
concentration or electron flux suggesting that one of the species
with g ) 2.00, characteristic of radicals which exhibit small g
is not a mechanistic precursor of another.
anisotropy due to minor spin-orbit coupling. The presence of
In summary, strong EPR signals, including a radical signal,
this signal is significant because it is the first observation of a
are elicited from an altered form of the nitrogenase MoFe protein
radical in a nitrogenase turnover sample. There are three pos-
when incubated under turnover conditions in the presence of C2H2.
sibilities for the origin of the radical species: (1) a C2H2 reduction
The identification of such signals under these conditions is an
intermediate, (2) an amino acid, perhaps one that is located along
important advance in our attempt to determine where and how
the electron-transfer path between the Fe protein docking site and
substrates become bound to the active site during nitrogenase
the FeMo-cofactor, and (3) the homocitrate molecule that provides
turnover, and to determine how the FeMo-cofactor polypeptide
the bidentate ligands to the Mo atom of the FeMo-cofactor. The
environment contributes to that process.
third signal in Figure 1 is a minor shoulder at g ) 1.97. Theorigin of this inflection is not clear and will not be further
9630127; D.R.D.) and USDA (96-35305-3730; B.J.H.) for support. We
To investigate the possible association of C2H2 with either of
also thank the Pennington Biomedical Research Center (Baton Rouge)
the first two signals, turnover samples were prepared with 13C2H2
for the use of their EPR spectrometer.
or C2D2 as substrate. The line widths of individual inflectionsare listed in Table 1 and reveal small yet reproducible isotope
Supporting Information Available: Figure 3 showing the g ) 2.12
effects. An overall general trend is evident; the line widths of
inflection of enzymatic turnover for R-H195Q (PDF). This material is
the g 2.12 signal (i.e., g ) 1.98 inflection) observed in C2D2
available free of charge via the Internet at http://pubs.acs.org.
turnover samples is narrower than what is observed for C2H2
turnover samples. Since the magnetogyric ratio for a deuteron issmaller (∼1/6) than for a proton, the EPR signals from species
(14) Shen, J.; Dean, D. R.; Newton, W. E. Biochemistry 1997, 36, 4884-
containing C2D2 adducts should be narrower than when C2H2 is
the substrate. This result is consistent with a broadening of the g
(15) Cameron, L. M.; Hales, B. J. Biochemistry 1998, 37, 9449-9456.
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