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Selenol Binds to Iron in Nitrogenase Iron-Molybdenum Cofactor: an Extended X-Ray Absorption Fine Structure Study (Azotobacter Vinelandii/GNXAS) STEVEN D

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Selenol Binds to Iron in Nitrogenase Iron-Molybdenum Cofactor: an Extended X-Ray Absorption Fine Structure Study (Azotobacter Vinelandii/GNXAS) STEVEN D Proc. Nati. Acad. Sci. USA Vol. 91, pp. 1290-1293, February 1994 Chemistry Selenol binds to iron in nitrogenase iron-molybdenum cofactor: An extended x-ray absorption fine structure study (Azotobacter vinelandii/GNXAS) STEVEN D. CONRADSON*t, BARBARA K. BURGESSO§, WILLIAM E. NEWTON, A. Di Cicco II, A. FILIPPONI**, Z. Y. Wutt, C. R. NATOLItt, BRITT HEDMAN**, AND KEITH 0. HODGSON*§# *Department of Chemistry,.Stanford University, Stanford, CA 94305; *Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92717; IDepartment of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061; IIDipartimento di Matematica e Fisica, Universita degli Studi di Camerino, Via Madonna delle Carceri, 63032 Camerino, Italy; **Dipartimento di Fisica, Universit& degli Studi deil'Aquila, Via Vetoio, 67010 Coppito-L'Aquila, Italy; ttlstituto Nazionale di Fisica Nucleare, Laboratori Nazionali di Frascati-C.P. 13, 00044 Frascati, Italy; and #Stanford Synchrotron Radiation Laboratory, Stanford University, SLAC, M.S. 69, P.O. Box 4349, Stanford, CA 94309 Communicated by Richard H. Holm, November 5, 1993 (receivedfor review August 11, 1993) ABSTRACT The biological N2-flxation reaction is cata- site-directed mutagenesis studies (11-13). This prediction lyzed by the enzyme nitrogenase. The metal cluster active site was confirmed by recent x-ray crystallographic results (14- of this enzyme, the iron-molybdenum cofactor (FeMoco), can 16), which showed that aCys-275 is a ligand of one of the Fe be studied either while bound within the MoFe protein com- atoms in FeMoco. ponent of nitrogenase or after it has been extracted into FeMoco contains seven Fe atoms but only one Mo atom (4, N-methylformamide. The two species are similar but not 5, 14, 15). The first definitive structural evidence that Mo and identical. For example, the addition ofthiophenol or selenophe- Fe were involved in an unprecedented polynuclear metal nol to isolated FeMoco causes its rather broad S 3/2 electron cluster came from extended x-ray absorption fine structure paramagetic resonance signal to sharpen and more closely (EXAFS) analysis at the Mo-K edge (17, 18). X-ray absorp- approach the signal exhibited by protein-bound FeMoco. The tion spectroscopy was also used to probe for chemical nature of this thiol/selenol binding site has been investigated reactivity (19) and electronic structural changes at the Mo site by using Se-K edge extended x-ray absorption frne structure (20), but little evidence of any significant effects or direct (EXAFS) to study selenophenol ligated to FeMoco, and the involvement ofMo was observed in these studies. Fe-K edge results are reported here. EXAFS data analysis at the ligand EXAFS was used to confirm the results obtained from the Se-K edge was performed with a set of software, GNXAS, that Mo-K studies and provided evidence for longer-range inter- provides for direct calculation ofthe theoretical EXAFS signals actions in FeMoco (21-23). and least-squares fits to the experimental data. Data analysis Insight into the complete structure of FeMoco has come results show definitively that the selenol (and by inference thiol) from the work of Rees and collaborators (14, 15). In this binds to Fe at a distance of 2.4 A. In contrast, unacceptable fits seminal study, they determined a model for protein-bound are obtained with either Mo or S as the liganded atom (instead FeMoco derived from the protein crystallography electron of Fe). These results provide quantitative details about an density map, currently at a resolution of 2.2 A. This model exchangeable thiol/selenol binding site on FeMoco in its iso- reveals a structure for FeMoco that consists of two subclus- lated, solution state and establish an Fe atom as the site of this ters (FeS3Fe3 and Fe3S3Mo), which are bridged by two S reaction. Furthermore, the utility of ligand-based EXAFS as a atoms and one additional ligand whose identity has not yet probe of coordination in polynuclear metal clusters is demon- been definitively established as published but that currently strated. is believed to be S. The only endogenous protein ligand that coordinates FeMoco through S is aCys-275, which binds to The biological N2-fixation reaction is catalyzed by the en- a single Fe atom at the terminus of the FeS3Fe3 subcluster. zyme nitrogenase. The active site of this enzyme is a metal The only additional ligands to FeMoco in the current model cluster of stoichiometry Mo:Fe7:.S29:homocitrate, which is (14, 15) are aHis-442 and homocitrate, both ofwhich bind to commonly designated as the iron-molybdenum cofactor Mo to complete its hexacoordination. (FeMoco) (1-3). FeMoco can be studied either when it is For isolated FeMoco, 19F NMR experiments that com- inside the MoFe protein component of nitrogenase or after it pared FeMoco, to model complexes have provided indirect has been extracted into the solvent N-methylformamide evidence for thiol binding to Fe (8, 9), although there is no (NMF) (1, 4, 5). The two species are similar but not identical specific evidence that it binds to the same Fe atom as (4, 5). In 1978, Rawlings et al. (6) reported that the addition aCys-275. Indirect evidence for thiol binding at Fe was also ofthiophenol to isolated FeMoco caused its S = 3/2 electron provided by the observation from EXAFS at the Mo-K edge. paramagnetic resonance (EPR) signal to sharpen and resem- In that study, it was shown that changes in ligation, which ble more closely the signal exhibited by protein-bound Fe- amounted to an increase in S coordination number at Mo of Moco. Subsequently, quantitative EPR (7) and 19F NMR <1 when FeMoco was treated with thiophenol or selenol, did [using p-CF3C6H4S- and p-CF3C6H4Se' (PiSoe) as the re- not originate from added thiol/selenol. If selenol were pres- porter ligands (8, 9)] have demonstrated that the thiol binds ent, it would have been readily detectable because ofits much specifically and reversibly to a single site on FeMoco. These stronger EXAFS backscattering behavior (18). data, combined with sequence comparisons (10), suggested In the work described below, we present a series of strongly that the a-subunit residue, aCys-275, might be the experiments that examine ligand binding to isolated FeMoco. single cysteine ligand to iteMoco in the MoFe protein. The ligation of FeMoco by a cysteinyl was further supported by Abbreviations: FeMoco, nitrogenase iron-molybdenum cofactor; EXAFS, extended x-ray absorption fine structure; NMF, N-methyl- formamide. The publication costs of this article were defrayed in part by page charge tPresent address: Los Alamos National Laboratory, M.S. D429, payment. This article must therefore be hereby marked "advertisement" MEE-11, Los Alamos, NM 87545. in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 1290 Downloaded by guest on September 27, 2021 Chemistry: Conradson et al. Proc. Natl. Acad. Sci. USA 91 (1994) 1291 Selenium (substituted for S in benzenethiol) was used as a For comparison, data were also analyzed by a more "reporter" ligand by examining the Se-K edge EXAFS. In conventional approach using empirically derived phase and the case ofuncoordinated selenol, one would see only carbon amplitude parameters (37, 38). Phase and amplitude param- and solvent (or perhaps counterion ifthere were a contact ion eters were derived as described (37, 38) from the model pair) contributing to the EXAFA signal. If (and only when) compounds listed (see above). However, a model suitable for strong selenol binding occurred to FeMoco (as established by extracting reliable Se-Mo parameters could not be found. In the NMR studies referenced above), a very significant per- the fits using the empirical parameters, the distance and turbation to the Se EXAFS would be expected. The EXAFS coordination number were allowed to vary. Results from could then be interpreted in terms of the nature and metric C-Se-X fits to these EXAFS data using the empirical Se-C details of the bound complex. This EXAFS study, as pre- and Se-Fe amplitude and phase parameters gave results sented below, provides unequivocal evidence that the thiol similar (1.5 C atoms at 1.92 A and 1.4 Fe atoms at 2.42 A from binding site on isolated FeMoco is to an Fe atom and not to Se) to those found for the GNXAS study whose results are Mo. A very preliminary account of this work, using a less described in more detail in the next section. Additional data complete EXAFS analysis, has appeared (24, 25). were measured and analyzed for a reduced sample with a PhSe-/FeMoco ratio of 2.4 and for two oxidized samples with a ratio of0.8 and 2.4, respectively (data not shown). The MATERIALS AND METHODS fit results were 1.2-1.5 C at 1.92-1.94 A and 0.67-1.15 Fe at Sample Preparations. FeMoco was isolated and concen- 2.42-2.44 A. C-Se-S fits gave chemically unreasonable re- trated as published (26) to give 1.91 ± 0.07 mM Mo, with a sults, whereas fits including Mo could not be tested because specific activity of 234 ± 12 nmol of C2H4 formed per min per of a lack of suitable model compounds. For this reason, the ng atom of Mo, after reconstitution of a FeMoco-deficient empirical parameter fits will not be considered further except MoFe protein from a mutant Azotobacter vinelandii strain. to point out that they are entirely consistent with the GNXAS This activity remained within 10o after the experiment. results discussed below. Semireduced FeMpco was produced by addition of10 electron equivalents of a neutralized, aqueous solution of Na2S204 (Eastman) just prior to data collection.
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