New Method for the Spin Quantitation of [4Fe−4S]+ Clusters with S

13632

J. Phys. Chem. B 2007, 111, 13632-13637

New Method for the Spin Quantitation of [4Fe-4S]+ Clusters with S ) 3/2. Application to the FS0 Center of the NarGHI Nitrate Reductase from Escherichia coli Pascal Lanciano,‡ Adrien Savoyant,‡ Ste´ phane Grimaldi,‡ Axel Magalon,§ Bruno Guigliarelli,‡ and Patrick Bertrand*,‡ Unite´ de Bioe´ nerge´ tique et Inge´ nierie des Prote´ ines (UPR9036) and Laboratoire de Chimie Bacte´ rienne (UPR9043), Institut de Biologie Structurale et de Microbiologie, CNRS, and Aix-Marseille UniVersite´ , 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France ReceiVed: July 5, 2007

In conventional analyses of g ≈ 5 signals given by [4Fe-4S]+ clusters with S ) 3/2, the effective g values that cannot be measured in the electron paramagnetic resonance (EPR) spectrum are deduced from rhombograms calculated by assuming that the g˜ matrix is isotropic with gx ) gy ) gz ) 2.00. We have shown that when the two low-field peaks corresponding to the Kramers doublets are visible in the spectrum, a new, independent piece of information about the system can be obtained by studying the temperature dependence of the ratio of the area under these peaks. By applying this method to the g ≈ 5 signals displayed by NarGHI nitrate reductase, we were able to determine all the parameters of the spin Hamiltonian of FS0 centers with S ) 3/2 and to measure accurately their number. Our results indicate that simple analyses based on the assumption of an isotropic g˜ matrix can give rise to very large errors.

Metal centers play a major role in biological systems. They are used as active sites and redox centers in enzymes and as binding sites in transport systems, and they participate in regulation mechanisms. Among the various techniques used to determine their stoichiometry and their redox properties, electron paramagnetic resonance (EPR) spectroscopy has played a major role. However, specific methods must be developed to study quantitatively new metal centers or conventional centers modified by site-directed mutagenesis which display unusual EPR signatures. A typical example is provided by [4Fe-4S]+ clusters with a S ) 3/2 ground state giving EPR signals in the g ≈ 5 region, which have been found in ferredoxins,1-4 enzymes,5-22 and very recently in a photosynthetic reaction center23 and a [Fe-Fe] hydrogenase maturation protein.24 Although some of these clusters are coordinated with three Cys and either His, Ser, or Asp,2,8,19 others are coordinated with the standard motif of four Cys residues.11,12,21 Very often, only a fraction of the clusters are in the S ) 3/2 form, the rest of the centers being present in the usual S ) 1/2 form. This variability reflects the great sensitivity of the magnetic properties of [4Fe-4S]+ clusters to the various factors that determine their spin coupling scheme: antiferromagnetic interactions, valence delocalization, and vibronic coupling.25 Although some theoretical models predict that the redox potential of [Fe-S] clusters may depend on these factors,25 the available data indicate that the potentiometric and functional properties of [4Fe-4S]+ clusters with S ) 3/2 and S ) 1/2 ground states do not differ significantly.3,10 The main part of the very anisotropic EPR spectrum given by [4Fe-4S]+ centers with S ) 3/2 is often masked by signals arising from S ) 1/2 species, and only one or two peaks are usually observed in the g ≈ 5 region. In this case, the spin * Corresponding author. Phone: (+00 33) 491164447. Fax: (+00 33) 491164578. E-mail: [email protected]. ‡ UPR9036. § UPR9043.

quantitation of the spectrum is carried out by comparing the area under one of these peaks with the integrated intensity of the spectrum given by a standard. This comparison requires the knowledge of the three effective g values. In all studies carried out so far on [4Fe-4S]+ clusters with S ) 3/2, these values were calculated by assuming that the g˜ matrix is isotropic, generally with gx ) gy ) gz ) 2.0.1,5,7,11,22 Despite the wellknown anisotropy of the g˜ matrix of [4Fe-4S]+ clusters with S ) 1/2, the validity and the accuracy of this procedure have never been questioned. The need for accurate methods to carry out the spin quantitation of [4Fe-4S]+ clusters with a S ) 3/2 ground state is well-illustrated by the case of the enzyme NarGHI nitrate reductase from Escherichia coli. This complex enzyme catalyzes the two-electron reduction of nitrate to nitrite when the bacterium is grown anaerobically in the presence of nitrate. Electrons provided by either ubiquinol or menaquinol are given to the membrane-bound NarI subunit which contains two b-type hemes and are transferred to the Mo-bisMGD cofactor of the NarG catalytic subunit through the four [Fe-S] centers (one [3Fe-4S]1+,0 cluster and three [4Fe-4S]2+,1+ clusters) of the NarH subunit. Prior to the determination of the X-ray crystal structure of NarGHI, a great amount of information about the coordination scheme and the redox properties of the metal centers had been obtained through a combination of site-directed mutagenesis and potentiometric experiments monitored by EPR spectroscopy.26 The crystal structure has provided a detailed picture of the electron-transfer chain made of the four [Fe-S] centers of NarH, which are now labeled as (starting from the catalytic subunit) : FS1, FS2, FS3, and FS4 ([3Fe-4S] cluster).27,28 This structure has also revealed the existence of an additional [4Fe-4S] cluster called FS0 in the NarG subunit.27,28 Although the presence of this cluster was expected on the basis of sequence comparisons with the catalytic subunits of other Mo-bisMGD-containing enzymes, it was not detected in previous EPR experiments.26

10.1021/jp075243t CCC: $37.00 © 2007 American Chemical Society Published on Web 11/08/2007

Spin Quantitation of [4Fe-4S]+ Clusters In a recent study, Rothery and co-workers have observed new signals at g ) 5.0 and g ) 5.6 in the low-temperature EPR spectrum displayed by NarGHI. These signals, which were found to titrate with Em ) -55 mV at pH 8.0, were ascribed to a [4Fe-4S]+ cluster with a S ) 3/2 ground state which was tentatively assigned to FS0.29 However, this assignment raises several points. Although FS0 is present in apomolybdo-NarGHI, the g ≈ 5 signals were not detected in this form of the enzyme.29 Moreover, no spin quantitation was attempted, and the signals due to the S ) 3/2 centers were implicitly assumed to be stoichiometric. In this work, we present a new and accurate method to carry out the spin quantitation of EPR signals given by S ) 3/2 species and we apply it to the g ≈ 5 signals displayed by NarGHI nitrate reductase. Our results indicate that the simple analysis based on the assumption of an isotropic g˜ matrix can give rise to very large errors. Materials and Methods Bacterial Strains and Plasmids. An E. coli nar-deficient strain, LCB306430 (nar25(narGH), narZ::Ω, ∆nap, thi-1, leu6, thr-1, rpsL175, lacY, KanR, SpcR and TP100031 (MC4100 ∆(mobAB), KanR), were used as a host for the experiments described herein. NarGHI was expressed from plasmid pVA700 (AmpR), which encodes the narGHJI operon under the control of the tac promoter.32 Another plasmid used in this work was pVA700-NarGH50SHJI33 encoding NarGH50SHI. Site-directed mutagenesis was performed on the pVA700 plasmid using the Expand PCR system of Roche as previously described33 yielding the pVA700-NarGC54SHJI and pVA700-NarGC58SHJI plasmids. The apomolybdo form of the enzyme was obtained as previously described.34 Growth Conditions and Preparation of Membrane Fractions. Cells were grown semi-anaerobically in a 2.5 L Erlenmeyer containing Terrific Broth medium at 37 °C.35 Enzyme overexpression was obtained by addition of 0.2 mM isopropyl 1-thio-β-D-galactopyranoside (IPTG). When appropriate, ampicillin (100 µg mL-1), spectinomycin (50 µg mL-1), and kanamycin (50 µg mL-1) were included in the growth medium. Cells were harvested and washed, and membranes were prepared by French pressure cell lysis and differential centrifugation.36 Enriched inner membrane vesicles were prepared from these crude membranes by sucrose step centrifugation.37 Membrane vesicles were suspended in 100 mM MOPS, 5 mM EDTA at pH 7.5 and stored at -80 °C until used. Membrane preparations were assayed for protein concentration by the method of Lowry et al.38 Immunological quantitation of NarGHI was achieved using rocket immunoelectrophoresis as described in ref 39. The NarGHI concentration was estimated to be in the 80-200 µM range, depending on preparations. Redox Potentiometry and EPR Spectroscopy. Redox titrations were performed anaerobically as described in ref 40. Redox potentials were adjusted with small additions of 100 mM sodium dithionite and measured with a combined Pt-Ag/AgCl/ KCl (3M) microelectrode in the presence of the same mediators (10 µM) as those listed in ref 40. Stable potentials were achieved in a few minutes, and samples were anaerobically transferred into calibrated EPR tubes which were rapidly frozen. All quoted potentials are given with respect to the standard hydrogen electrode. EPR measurements were performed on a Bruker Elexsys E500 spectrometer equipped with a standard rectangular Bruker cavity (ST4102) which was fitted to an Oxford Instruments Helium flow cryostat (ESR900). The g ≈ 5 part of the spectrum was recorded in nonsaturating conditions between 3

J. Phys. Chem. B, Vol. 111, No. 48, 2007 13633 and 15 K. The areas under the g ) 5.0 and g ) 5.6 peaks were evaluated by simulating the signal as the sum of two Gaussian components whose amplitudes and line widths were adjusted independently at each temperature. Spin quantitations were performed under nonsaturating conditions using 1 mM Cu(EDTA) as a standard. Model. The main features of the standard model describing the EPR properties of a S ) 3/2 system are briefly recalled. The spin Hamiltonian is written

HS ) D(SZ2 - S(S + 1)/3) + E(SX2 - SY2) + β(gxSxBx + gySyBy + gzSzBz) where D and E are the zero-field splitting parameters and gx, gy, and gz the principal components of the g˜ matrix. The degeneracy of the {|3/2, MS〉} spin states is partly removed by the zero-field splitting terms, which gives rise to two Kramers doublets separated by ∆ ) 2|D|(1 + 3R2)1/2, where R ) E/D. The degeneracy of the doublets is removed in turn by the Zeeman terms. If the condition hν 3/2 could be detected in the low-field part of the spectrum using either parallel- or perpendicular-mode EPR spectroscopy. Temperature Dependence of the Signals. The temperature dependence of the areas under the g ) 5.6 and g ) 5.0 peaks was quantitatively studied between 3 and 15 K. To remove the contribution of adventitious iron at g ) 4.3 and of high-spin hemes present in the membrane preparations (Figure 1A), these areas were determined by using the simulation procedure described in Materials and Methods. The line widths were found to be essentially constant between 3 and 7 K, but a spin-lattice relaxation broadening was observed above 9 K. Although the temperature dependence of the area was found to be qualitatively similar to that reported for the peak amplitudes,29 the relaxation broadening makes the area decrease much more slowly than the amplitude when the temperature is increased. This effect must of course be considered in any quantitative study of the intensity. Our data show unambiguously that the g ) 5.6 and g ) 5.0 signals arise from the ground (k ) 1) and excited (k ) 2) doublet, respectively. Thus, the zero-field splitting parameter D is negative. The temperature dependence of the g ) 2.79 signal was found to correlate with that of the g ) 5.0 peak (data not shown), showing that it comes from the same doublet. On the basis of this assignment, eq 2 should read for the two doublets:

A1 ∝ N/(1+ exp(-∆/kBT))gP1D1P1/2th(hν/2kBT) A2 ∝ N exp(-∆/kBT)/ (1 + exp(-∆/kBT))gP2D2P1/2th(hν/2kBT) (3) where N is the total number of S ) 3/2 species. From these equations, we deduce

A1/A2 ) (gP1D1/gP2D2) exp(∆/kBT)

(4)

Plotting ln(A1/A2) as a function of T yields (Figure 2):

∆ ) 4.4 ( 0.2 cm-1; gP1D1/gP2D2 ) 0.15 ( 0.01 The quoted errors were deduced from the slope and the Y-axis intercept of limiting acceptable lines. The large value of ∆ compared to hν ) 0.3 cm-1 ensures the validity of the perturbation treatment used to derive the expressions of the effective g values. To determine N by comparing the area under the low-field peaks with the integrated intensity of a standard, the factors gP1D1 and gP2D2 involved in eqs 3, which depend on the effective g values and therefore on R ) E/D, must be evaluated. Determination of the Effective g Values. The effective g values of S ) 3/2 centers are generally calculated by assuming that the g˜ matrix is isotropic with gx ) gy ) gz ) 2.0, which

Spin Quantitation of [4Fe-4S]+ Clusters

J. Phys. Chem. B, Vol. 111, No. 48, 2007 13635

gP1D1 ) β(g1z)2[(g1x)2 + (g1y)2]/ {8hν[(g1z)2 - (g1x)2]1/2[(g1z)2 - (g1y)2]1/2} gP2D2 ) β(g2y)2[(g2x)2 + (g2z)2]/ {8hν[(g2y)2 - (g2x)2]1/2[(g2y)2 - (g2z)2]1/2} (5) together with the following set of data:

g1z ) 5.60, g2y ) 5.00, g2x ) 2.79, gP1D1/gP2D2 ) 0.15

Figure 2. Plot of the natural logarithm of the ratio of the area under the g ) 5.6 and g ) 5.0 peaks against the reciprocal of the temperature. The areas were determined as described in Materials and Methods. Data points were fitted to eq 3 in the text.

It turns out that the ratio gP1D1/gP2D2 is much more sensitive to R than to {gx, gy, gz,}. Therefore, an approximate value of R is obtained by plotting gP1D1/gP2D2 calculated from eqs 5 with gx ) gy ) gz ) 2, as a function of R. This gives R ) 0.183 (Figure 4). From this value and those of g1z, g2y, g2x, a first set {gx, gy, gz,} is deduced. When this set is used in eqs 5, a new curve giving gP1D1/gP2D2 as a function of R is obtained, which yields a slightly different R value. This iterative procedure converges rapidly to (Figure 4)

R ) 0.179, gx ) 1.935, gy ) 2.026, gz ) 1.924 From the values of R and ∆, we deduce

D ) -2.0 cm-1, E ) -0.36 cm-1 Although the main components of the g˜ matrix of S ) 1/2 and S ) 3/2 spin states can be different, it is interesting to note that the values deduced from this analysis are similar to those of [4Fe-4S]+ clusters with S ) 1/2. To our knowledge, this is the first time that these components are determined for a [4Fe4S]+ cluster with S ) 3/2. The values of gkx, gky, gkz (k ) 1, 2) calculated with this set of g values are plotted as a function of R in Figure 3 (solid lines). Using R ) 0.179, one obtains the effective g values of the Kramers doublets of FS0:

g1x ) 1.08, g1y ) 0.948, g1z ) 5.60 g2x ) 2.79, g2y ) 5.00, g2z ) 1.75

Figure 3. Plot of the effective g values for the two Kramers doublets of a S ) 3/2 system as a function of E/D. They were calculated from eqs 1 by assuming either an isotropic g matrix with gx ) gy ) gz ) 2.0 (dashed lines) or an anisotropic one with gx ) 1.935, gy ) 2.026, gz ) 1.924 (solid lines).

enables gkx, gky, gkz to be plotted as a function of the single parameter R ) E/D.5 These plots are often called “rhombograms” in the literature.44 Applying this method to the g values 5.60, 5.00, and 2.79 displayed by FS0 leads to the assignments g1z ) 5.6 with R ) 0.28, and g2y ) 5.0, g2x ) 2.8 with R ) 0.19 (Figure 3, dashed lines). The significant discrepancy between the R values obtained for the two doublets means that the assumption gx ) gy ) gz ) 2 is wrong. Therefore, eqs 1 giving the effective g values in terms of the four parameters {gx, gy, gz,, R} must be used. These four parameters can be determined by using an iterative procedure based on these expressions and on the following equations:41

Spin Quantitation of the S ) 3/2 Species. Since the concentration of the NarGHI enzyme present in the membrane preparation cannot be accurately measured, the amount of S ) 3/ species was determined by comparing the area A and A 2 1 2 (eqs 3) of the low-field peaks displayed at g ≈ 5 by a sample poised at -250 mV with the intensity of the EPR spectrum displayed by the [3Fe-4S]+ centers of the enzyme. This intensity I0 was obtained by double integration of the spectrum given by a sample poised at +307 mV, in which all [3Fe-4S] clusters are oxidized. This intensity is proportional to

I0 ∝ N0(gP0)avP01/2th(hν/2kBT0) The average intensity factor (gP0)av 41 calculated with the values g| ) 2.02 and g⊥ ) 1.98 measured in the spectrum is equal to 0.50. The ratio N/N0 deduced from the comparison of I0 measured at T0 ) 12.5 K with A1 and A2 measured at various temperatures is given in Figure 5. From these data, it is concluded that N/N0 ) 0.40 ( 0.03. The main source of error comes from the value of the parameters gP1D1/gP2D2 and ∆ which were deduced from Figure 2, and therefore from the scattering of the values of the area A1 and A2 which is also apparent in Figure 5. It is worth recalling that these areas were evaluated independently at each temperature, by leaving the amplitude and the line width of the Gaussian lines as adjustable

13636 J. Phys. Chem. B, Vol. 111, No. 48, 2007

Lanciano et al. TABLE 1: Intensity Factors gPD of the Low-Field Peaks of the Two Kramers Doubletsa gP1D1 gP2D2

this worka

rhombograms

0.40 2.6

0.93 b 2.7 c

a These numbers, expressed in tesla-1, were calculated from eqs 1 and eqs 5 in the text, with ν ) 9.469 × 109 Hz and (a) gx ) 1.935, gy ) 2.026, gz ) 1.924, and R ) 0.179; (b) gx ) gy ) gz ) 2 and R ) 0.28; and (c) gx ) gy ) gz ) 2 and R ) 0.19.

Figure 4. Plot of the ratio gP1D1/gP2D2 as a function of E/D given by eqs 5 by assuming either an isotropic g matrix with gx ) gy ) gz ) 2.0 (dashed lines) or an anisotropic one with gx ) 1.935, gy ) 2.026, gz ) 1.924 (solid lines).

Figure 5. Plot of the ratio of the number of S ) 3/2 species over the number of [3Fe-4S]+ clusters. This ratio was deduced from the comparison of the intensity of the [3Fe-4S]+ signal measured at 12.5 K with the area under the g ) 5.6 (filled squares) and g ) 5.0 (open circles) peaks measured between 3 and 15 K.

parameters. This procedure is expected to increase the accuracy of the numbers deduced from a least-squares analysis of the data. Discussion The spin quantitation of EPR spectra given by paramagnetic species with S > 1/2 is never a straightforward issue. A major point is that the amplitude of their spectral features is much reduced by comparison with those displayed by S ) 1/2 centers. Indeed, the large anisotropy of the effective g values acts in two different ways: the pattern of resonance lines is spread over a wide field range, and the line width increases dramatically when the resonance line is shifted upfield in field-swept spectra. As a result, the high-field resonance lines are hardly detected. By comparison with S ) 1/2 centers, an obvious advantage of species with S > 1/2 is that they can give rise to more than one transition, but this advantage is rarely fully exploited. The present work illustrates how this can be done in the case of [4Fe-4S]+ clusters with S ) 3/2. In all previous quantitative studies carried out on these clusters, the effective g values needed in the calculation of the transition probabilities and of the quantities gPD (eqs 5) were deduced from rhombograms calculated by assuming gx ) gy ) gz ) 2.0 1,5,7,11,22 or by using other values like 1.98,2,4,6 2.08,6 or 1.873 18 without justification. In the case of the FS0 center of NarGHI, we were able to determine accurately the complete

set of effective g values of the two doublets without relying on any assumption, by using the g values of the two low-field peaks and of a derivative-shaped feature, together with the quantity gP1D1/gP2D2 deduced from the temperature dependence of the area under the two peaks. The accuracy of the simple method based on rhombograms can therefore be evaluated. The quantities gPkDk (k ) 1, 2) calculated with this method are compared with those obtained in the present work in Table 1. Although the method based on rhombograms gives a correct result when applied to the g2y ) 5.0 peak, it overestimates gP1D1 and therefore underestimates the number N of S ) 3/2 centers by a factor equal to 2.3 when applied to the g1z ) 5.6 peak. This is because the curve giving g1z as a function of R is rather flat (Figure 3) so that a slight deviation of gz from 2.00 can greatly alter R and therefore the two other effective g values. Thus, the analysis of the intensity of the peak given by doublet 1 based on rhombograms is not expected to give reliable results. Our study shows conclusively that the g ≈ 5 signals are due to FS0 centers with a S ) 3/2 ground state and that these centers represent 40% of the FS0 centers present in the native enzyme. This proportion can be evaluated for other forms of the enzyme by comparing the area under the g ≈ 5 peaks and the integrated intensity of the [3Fe-4S]+ centers measured in this form to those measured in the native enzyme. In this way, the proportion of FS0 centers with S ) 3/2 was found to be equal to 30 and 40% in the apomolybdo and the NarGH soluble forms, respectively. The fact that the g ≈ 5 signals are substoichiometric is not surprising, since this is generally the case for [4Fe4S]+ clusters with S ) 3/2. However, our results imply that 60% of the FS0 centers are present in another spin state. Since no signal arising from centers with S > 3/2 could be detected in the low-field part of the spectrum using either parallel- or perpendicular-mode EPR spectroscopy, the most likely hypothesis is that these centers are in the S ) 1/2 form. They are therefore expected to give a component which titrates with Em8 ≈ -55 mV in the g ≈ 2 region of the spectrum. The spectral changes which occur in this range of potentials consist of the appearance of a small peak at g ≈ 1.97, of shoulders in the g ≈ 1.93 region of the spectrum, and of very broad lines at g ≈ 2.08 and g ≈ 1.82, which give rise to an increase of the total intensity of the spectrum corresponding to about one center per molecule. These very peculiar changes were observed in redox titrations carried out on various forms of the enzyme: heatreleased NarGH,32 NarGH soluble fraction,32 and membrane bound NarGHI.45 They were originally attributed to the reduction of FS3 in an indirect way. In the case of the FS1, FS2, and FS4 centers, the correspondence between the EPR signals, the redox potentials, and the motif of coordinating cysteines was deduced from site-directed mutagenesis experiments in which the replacement of a coordinating cysteine with another residue gave rise to spectral changes showing unambiguously that a specific cluster was either loss or converted into a [3Fe-4S]1+,0 cluster.26,32,39,46,47 However, this method failed in the case of FS3 because mutating the coordinating cysteines resulted either in the loss of the four [Fe-S] clusters or in EPR spectra which

Spin Quantitation of [4Fe-4S]+ Clusters did not differ significantly from those of the wild type enzyme.32,39 Thus, the spectral changes observed in the intermediate range of potentials were attributed to the reduction of FS3 because it was the last [4Fe-4S]2+,1+ cluster of NarGHI known at that time. Given the fact that 60% of the FS0 centers are expected to give a component which appears in this range of potential in the g ≈ 2 region of the spectrum, it appears that new, detailed redox titrations followed by EPR are needed to differentiate the signals and the redox potentials of the FS0 and FS3 centers. The knowledge of the midpoint potential of all [Fe-S] centers of NarGHI nitrate reductase is an essential step toward the understanding of the catalytic cycle of the enzyme.48 Such experiments are under way in our laboratory. Conclusion In the present work, new information about the system made of the two Kramers doublets of a S ) 3/2 species was obtained by studying the temperature dependence of the ratio of the area under the two low-field peaks of their spectra. This procedure can obviously be applied to Kramers systems with S > 3/2. In the usual case where the main axes of the g˜ matrix and of the zero-field splitting terms can be considered as identical and the (2S + 1)/2 Kramers doublets are well-separated from each other, their EPR spectra depend on four unknown parameters: the three main values of the g˜ matrix and the ratio R ) E/D. The measurement of the g values of (2S + 1)/2 low-field peaks and the study of the temperature dependence of the [(2S + 1)/2-1] ratio of the area under these peaks provide 2S constraints to determine four parameters. Thus, the problem is overdetermined as soon as S g 5/2. When the separation between the Kramers doublets is small, the spectra depend on five unknown parameters and the preceding conclusion should hold true. However, in this case the situation is much more involved since a numerical simulation is needed. Acknowledgment. This work was supported by the CNRS, the Universite´ de Provence, and the Agence Nationale de la Recherche (Project PCV/ERMoE). P.L. was supported by a MRT fellowship. References and Notes (1) George, S. J.; Armstrong, F. A.; Hatchikian, E. C.; Thomson, A. J. Biochem. J. 1989, 264, 275. (2) Conover, R. C.; Kowal, A. T.; Fu, W. G.; Park, J. B.; Aono, S.; Adams, M. W.; Johnson, M. K. J. Biol. Chem. 1990, 265, 8533. (3) Park, J. B.; Fan, C. L.; Hoffman, B. M.; Adams, M. W. J. Biol. Chem. 1991, 266, 19351. (4) Duderstadt, R. E.; Brereton, P. S.; Adams, M. W.; Johnson, M. K. FEBS Lett. 1999, 454, 21. (5) Lindahl, P. A.; Day, E. P.; Kent, T. A.; Orme-Johnson, W. H.; Munck, E. J. Biol. Chem. 1985, 260, 11160. (6) Zambrano, I. C.; Kowal, A. T.; Mortenson, L. E.; Adams, M. W.; Johnson, M. K. J. Biol. Chem. 1989, 264, 20974. (7) Flint, D. H.; Emptage, M. H.; Finnegan, M. G.; Fu, W.; Johnson, M. K. J. Biol. Chem. 1993, 268, 14732. (8) Kowal, A. T.; Werth, M. T.; Manodori, A.; Cecchini, G.; Schroder, I.; Gunsalus, R. P.; Johnson, M. K. Biochemistry 1995, 34, 12284. (9) Koehler, B. P.; Mukund, S.; Conover, R. C.; Dhawan, I. K.; Roy, R.; Adams, M. W. W.; Johnson, M. K. J. Am. Chem. Soc. 1996, 118, 12391.

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