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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 UniVersity Department of Biochemistry,Virginia Polytechnic Institute and State UniVersity, Blacksburg, Virginia 24061 ReVised 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 Acknowledgment.
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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