Detection of a New Radical and FeMo-Cofactor EPR Signal during Acetylene Reduction by the r-H195Q Mutant of Nitrogenase Morten Sørlie, ² Jason Christiansen, ‡ Dennis R. Dean,* ,‡ and Brian J. Hales* ,² Department of Chemistry, Louisiana State UniVersity Baton Rouge, Louisiana 70803-1804 Department of Biochemistry,Virginia Polytechnic Institute and State UniVersity, Blacksburg, Virginia 24061 ReceiVed May 13, 1999 ReVised Manuscript ReceiVed August 19, 1999 The Mo-dependent nitrogenase of Azotobacter Vinelandii is a two-component system consisting of iron (Fe) protein and molybdenum-iron (MoFe) protein. In addition to the physiologi- cally relevant conversion of N 2 to NH 3 , nitrogenase catalyzes the ATP-dependent reduction of simple, multiple bonded molecules such as C 2 H 2 , HCN, and HN 3 . Substrate reduction is believed to take place at a MoFe 7 S 9 ‚homocitrate metal cluster called FeMo- cofactor which is contained within the R-subunit of the MoFe protein. During catalysis the Fe protein serves as a specific, MgATP-dependent reductant of the MoFe protein. In its as- isolated form the MoFe protein displays a rhombic S ) 3/2 EPR signal (g ) 4.3, 3.6, and 2.0) originating at the FeMo-cofactor. During turnover this signal is diminished by up to 90% to an EPR-silent state. When the potent noncompetitive inhibitor CO is present in the turnover system, two different intense S ) 1/2 are generated, 1-3 lo-CO (g ) 2.09, 1.97, 1.93; P CO ) 0.08 atm) and hi-CO (g ) 2.17, 2.06, 2.06; P CO ) 0.5 atm). Recently these signals have been investigated with ENDOR spectroscopy 4-7 and were shown to arise from one or two molecules of CO, respectively, bound to the FeMo-cofactor. Although minor substrate-induced EPR signals have been elicited from the MoFe protein under turnover conditions, 2,8 to date no nitrogenase substrates have been shown to induce strong signals for the wild- type enzyme similar to those observed when CO is present under turnover conditions. Herein, we describe the first report of intense EPR signals, including a radical signal, that are elicited from an altered form of the MoFe protein (R-H195Q) when incubated in the presence of C 2 H 2 under turnover conditions. The R-H195Q mutant form of the MoFe protein was con- structed, isolated, and investigated by Kim et al. 9 This altered form of the MoFe protein has glutamine substituted for the R-subunit histidine-195 residue, which is a strictly conserved amino acid within the MoFe protein and is within hydrogen- bonding distance of the FeMo-cofactor. 10-12 The altered protein has received much attention since it previously has been shown that, although its phenotype for reduction of most substrates resembles that of wild-type (acetylene has a nearly identical K m ), it has the unique property that N 2 binds during turnover but is not significantly reduced. 9,13 This mutant MoFe protein also appears to be minimally altered spectroscopically because it exhibits a rhombic S ) 3/2 EPR signal nearly identical with that found for the wild-type MoFe protein in the as-isolated state. Thus, it was worthwhile to determine if EPR signals arising from enzyme turnover events, previously unobservable in the wild- type MoFe protein, could be observed in this altered protein. Figure 1 shows the turnover-dependent, acetylene-induced EPR signal of R-H195Q MoFe protein in the g-2 region (sample was rapidly frozen in liquid N 2 3 min following initiation of turnover) with the full spectrum included as the insert. This signal (spin integration 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 not detected when the wild-type enzyme is used under the same conditions. The numerous inflections show that this signal originates from more than one paramagnetic species. To inves- tigate the relationship of these signals, turnover samples were prepared in the presence of C 2 H 2 and allowed to incubate at different temperatures (10, 30, and 45 °C) prior to rapid freeze- quench. Figure 2 shows the EPR spectra obtained from samples incubated at 30 and 45 °C illustrating that the g ) 2.00 inflection has smaller amplitude relative to the g ) 2.12 inflection at 45 °C compared to 30 °C. Similar changes were observed in turnover samples made at 30 °C compared to those at 10 °C (results not shown) indicating that the g ) 2.00 and 2.12 inflections represent different species. Temperature-dependent and power-dependency ² Louisiana State University. ‡ Virginia Polytechnic Institute and State University. (1) Yates, M. G.; Lowe, D. J. FEBS Lett. 1976, 72, 121-126. (2) Lowe, D. J.; Eady, R. R.; Thorneley, R. N. F. Biochem. J. 1978, 173, 277-290. (3) Davis, L. C.; Henzl, M. T.; Burris, R. H.; Orme-Johnson, W. H. Biochemistry 1979, 18, 4860-4869. (4) Pollock, R. C.; Lee, H.-I.; Cameron, L. M.; DeRose, V. J.; Hales, B. J.; Orme-Johnson, W. H.; Hoffman, B. M. J. Am. Chem. Soc. 1995, 117, 8686-8687. (5) Christie, P. D.; Lee, H.-I.; Cameron, L. M.; Hales, B. J.; Orme-Johnson, W. H.; Hoffman, B. M. J. Am. Chem. Soc. 1996, 118, 8707-8709. (6) Lee, H.-I.; Hales, B. J.; Hoffman, B. M. J. Am. Chem. Soc. 1997, 119, 11395-11400. (7) Lee, H.-I.; Cameron, L. M.; Hales, B. J.; Hoffman, B. M. J. Am. Chem. Soc. 1997, 119, 10121-10126. (8) Rasche, M. E.; Seefeldt, L. C. Biochemistry 1997, 36, 8574-8585. (9) Kim, C.-H.; Newton, W. E.; Dean, D. R. Biochemistry 1995, 35, 2798- 2808. (10) Kim, J.; Rees, D. C. Science 1992, 257, 1677-1682. (11) Kim, J.; Rees, D. C. Nature 1992, 360, 553-560. (12) Chan, M. K.; Kim, J.; Rees, D. C. Science 1993, 260, 792-794. (13) Dilworth, M. J.; Fischer, K.; Kim, C. H.; Newton, W. E. Biochemistry 1998, 37, 17495-17505. Figure 1. The g-2 region of the EPR spectrum of enzymatic turnover for R-H195Q in the presence of acetylene. Inflections originating from the g ) 2.12 signal (g ) 2.12, 1.98, 1.95) are marked (a), the g ) 2.00 signal is marked (b), and the g ) 1.97 signal is marked (c) The whole spectrum is shown as the insert. Note that the S ) 3/2 FeMo-cofactor signal is almost completely replaced by the S ) 1/2 signals. To obtain the high resolution observed in both spectra the modulation amplitude was 0.1 mT, well below the value normally used to record spectra of metal clusters. Experimental conditions: [Fe protein]/[R-H195Q] ) [0.020 mM]/[0.100 mM] ) 1:5; [C2H2] ) 0.1 atm; [ATP] ) 10 mM; [MgCl2] ) 25 mM; [Na2S2O4] ) 20 mM; 50 mM TES-KOH, pH 7.4. Spectrometer parameters: microwave frequency ) 9.45 GHz; microwave power ) 2 mW; modulation amplitude ) 0.1 mT; temperature ) 4K. 9457 J. Am. Chem. Soc. 1999, 121, 9457-9458 10.1021/ja991599+ CCC: $18.00 © 1999 American Chemical Society Published on Web 09/24/1999