The iron-porphyrin prosthetic group is typically the active site for heme proteins.

Paramagnetic heme iron states are commonly encountered, in particular the high-spin (S=5/2)

iron(III) form which is among the most difficult to study by NMR. Due to the five unpaired electrons

in this state, extremely large proton hyperfine shifts

occur (Figure 1). These resonances relax extremely quickly and produce a proton chemical shift

range that is typically > 100 ppm. Figure 1 illustrates the range of the hyperfine-shifted proton

resonances. It shows 1D 1H presaturation spectra of unliganded met Myoglobin in 90% H2O/D2O.

Figure 1 also illustrates one of the most vexing problems encountered in studying high spin

paramagnetic proteins, namely the extreme baseline distortion brought about by the combination

of large spectral widths and the comparatively intense diamagnetic envelope. The diamagnetic

envelope consists largely of nuclei with vastly longer relaxation times than the few hyperfine-

shifted resonances that lie within it. Our goal has been to develop pulse methods leading to

accentuated hyperfine shifted signals and baselines that do not need to be manipulated in post-

processing in order to facilitate hyperfine resonance assignment in these molecular systems.

Figure 1: Off-resonance presaturation 1D spectra of metMb in 90% H2O using A) 7.4 s 90 degree square read pulse and B) an 81.6 s 90 degree polychromatic pc17c pulse 6. Note the better baseline performance of the polychromatic pulse. 1024 scans were recorded for a total time of 39 minutes.

Most hyperfine proton resonance assignments are accomplished by studying the NOE

between protons of the Fe(III) ligands and working outward, further from the metal center. Rapid

relaxation of the hyperfine-shifted resonances poses restrictive problems for 2D NOESY

techniques which has led most researchers to use 1D difference steady state NOE methods 1,2.

These methods typically suffer greatly from subtraction artifacts, especially in 90% H2O/D2O

solution. Methods recently developed to address poor baseline and subtraction qualities are the

Super-WEFT 3 and PASE4 pulse-sequences. While these methods have advanced the study of

paramagnetic proteins, recent advances in pulse shaping offer the possibility of further improving

both the baseline characteristics as well as the quality of the subtraction in difference spectra.

Here we describe our efforts towards recording spectra of high-spin heme proteins with excellent

baselines and high quality difference spectra, particularly for proteins in 90% H2O/D2O solution,

which should allow for observation of NOE’s to exchangeable amide protons.

Samples of horse skeletal muscle metmyoglobin (Sigma) were 2 mM protein in D2O, 100

mM KH2PO4 pH 7.8 and 3 mM protein in 90% H2O/ D2O, 100 mM KH2PO4 100 mM KH2PO4 pH 7.8.

The recombinant truncated FixLH was expressed in E. coli, BL21DE3 and purified to homogeneity

by column chromatography (gel filtration and ion exchange). Samples for NMR were 2 mM in 20

mM Tris (Sigma), 100mM NaCl (Fisher), pH 8.0. Spectra were recorded on a Varian Inova 500

spectrometer operating at 499.843 MHz for proton utilizing a Nalorac triple resonance Z-axis

gradient probe. All spectra were recorded at 22 °C utilizing a spectral width of 100 KHz and

acquisition time of 80 ms. Spectra were processed using Varian’s VNMR software or MestReC5 , a

freeware processing package for PCs. FIDs were apodized with 20 Hz of exponential line

broadening prior to zero filling to 65K points and Fourier transformation. 1D difference spectra were

obtained by subtraction of the on-resonance irradiation FID with the off-resonance irradiation (-50

ppm) FID followed by apodization and Fourier transformation. Spectra were not baseline corrected

in any way beyond standard DC offset correction.

We chose metmyoglobin for sequence development because its proton hyperfine

spectrum is well known1,2. Some of these spectra are presented below. However the perfected

pulse sequences have allowed us to begin producing new data on the active form of metFixLH

from B. japonicum, a unique heme protein sensor (see further).

Figure 2: Various 1D methods for metMb in 90% H2O. In all spectra the carrier is placed at 40

ppm.

Spectrum A is the same as shown in figure 1b) utilizing the polychromatic pulse. 1024 scans

were recorded for a total time of 39 minutes.

Spectrum B was taken using the pulse sequence “SHWEFT” in Figure 3A which utilizes a

WURST-80 7 inversion pulse which inverts only the diamagnetic region and a PC17c read pulse.

The repetition rate was 6 s-1, the number of scans was 1024 for a total time of 2.8 minutes.

Spectrum C was taken with the “PASE” 4 pulse sequence of Figure 3B which utilizes strong

presaturation of the H2O and WALTZ 16 proton decoupling to effectively suppress the

diamagnetic region. The repetition rate was 3.6 s-1, the number of scans was 1024 for a total time

of 4.75 minutes.

Spectrum D was taken using the “SHWEFT-PASE” pulsesequence shown in Figure 3C which

replaces the presaturation of the water and uses the PC17c read pulse. The repetition rate was

3.9 s-1, the number of scans was 1024 for a total time of 4.38 minutes.

Figure 3: Pulse-sequences used for recording 1D spectra of metMb in D2O and 90% H2O solutions. The carrier was placed at 40 ppm for all sequences.

A) SHWEFT sequence: P1 = 4.6 ms WURST-80 band selective pulse shifted –18462 Hz from the carrier which is placed at 40 ppm. The maximum B1 field for the WURST-80 pulse was 1.7 KHz. The delays are as follows: d1 = 1.0 ms,1=20 ms, G1 = 1.0 ms @ 20 G/cm, 2=44 ms, acquisition time=80 ms. P2 = 81.6s PC17c pulse on resonance at a maximum B1 field of 41.9 KHz.1=0, 0, 0, 0, 1, 1, 1, 1, 2, 2, 2, 2, 3, 3, 3, 32=3=0,2, 1, 3, 1, 3, 2, 0, 2, 0, 3, 1, 3, 1, 0, 2

B) PASE sequence: presat of H2O with a 75 ms pulse on the H2O resonance at a B1 field of 135 Hz. P1 = 268 s square pulse while P2 = 7.4 s square 90 degree pulse. The delays are as follows: d1 = 20.0 ms, 1=2.0 s, 2=s, acquisition time=80 ms. WALTZ-16 decoupling=100 ms at a B1 field of 942 Hz.1=1, 2=3, 3=4=0, 2, 1, 3, WALTZ-16 phase=0, presat phase= 1, 1, 2, 2, 3, 3, 0, 0

C) SHWEFT-PASE sequence: P1 = 4.6 ms WURST-80 band selective pulse shifted –18462 Hz from the carrier which is placed at 40 ppm. The maximum B1 field for the WURST-80 pulse was 1.7 KHz. The delays are as follows: d1 = 1.0 ms, 1=20 ms, G1 = 1.0 ms @ 20 G/cm, t2=60 s, t3=2.0 s, t4=4.0 s, and the acquisition time was 80 ms. P2 = P3 = 268 s square 90 degree pulse, P4 = 81.6 s PC17c pulse on resonance at a maximum B1 field of 41.9 KHz. WALTZ-16 decoupling = 100 ms at a B1 field of 942 Hz.1=0, 0, 0, 0, 1, 1, 1, 1, 2, 2, 2, 2, 3, 3, 3, 32=1, 3=3, WALTZ-16=04=5=0, 2, 1, 3, 1, 3, 2, 0, 2, 0, 3, 1, 3, 1, 0, 2

D) SHWEFT-NOE sequence: P1 = 4.6 ms WURST-80 band selective pulse shifted –18462 Hz from the carrier which is placed at 40 ppm. The maximum B1 field for the WURST-80 pulse was 1.7 KHz. The delays are as follows: d1 = 1.0 ms, 1=20 ms, G = 0.5 ms @ 10 G/cm G2 = 1.0 ms @ 20 G/cm, 2 + saturation time = 64 ms and the acquisition time was 80 ms. P2 = 81.6 s PC17c pulse on resonance at a maximum B1 field of 41.9 KHz. CW saturation of the selected targets was at a B1 field of 170 Hz.1=0, 0, 0, 0, 1, 1, 1, 1, 2, 2, 2, 2, 3, 3, 3, 32=3=0, 2, 1, 3, 1, 3, 2, 0, 2, 0, 3, 1, 3, 1, 0, 2

Figure 4: 1D NOE difference spectra of 2 mM met Mb in D2O at 22 °C.A. 8-methyl resonance at 92.8 ppm (80 ms cw) at a B1 field of 170 Hz. Control irradiation at –50 ppm; 34,816 transients, 3 hrs total experiment duration.B. 1D difference NOE spectrum resulting from irradition of the heme 1-methyl resonance at 52.8ppm with same cond1D SHWEFT spectrum, 1024 transients.C. 1D difference NOE spectrum acquired with the pulse sequence shown in Figure 3D by irradiating the heme itions as in B.Note the very clear NOEs within the diamagnetic region that result from the excellent subtraction and baseline qualities of this pulse sequence.

Nitrogen fixation is one of the most important biological processes to occur in nature.

Initiation of nitrogen fixation is signaled by a protein that senses the partial pressure of O2 . This

protein, termed FixL is a 2-domain heme containing protein and it's role is to help control in vivo

expression of the nif/fix genes in plant root nodules7,8. FixLH (~12 kD), the recombinant heme

domain contains the Fe-protoheme IX sensor that initiates the signal with the loss of molecular

oxygen from the heme at low partial pressures of O2. A conformational change is presumed to

occur in FixL upon O2 dissociation, which is thought to activate the enzymatic domain. The

mechanism of the signal propagation is unknown at this time, although it occurs both with

deoxygenation in the Fe(II) state and with ligand loss in the Fe(III) state. In the absence of ligand,

the oxidized form of FixLH is high spin (Fe(III); S=5/2), with hyperfine shifts ranging over ~110

ppm. Assignment of the 1H resonances in the heme active site is imperitive to understanding the

structural changes that may occur upon ligand binding and dissociation.

Application of these pulse methods to the oxidized form (Fe3+) of truncated, recombinant

Bradyrhizobium japonicum (Bj) heme domain, FixLH, has resulted in unambiguous, complete

heme hyperfine resonance assignments. Examples of these connectivities, including the crucial

heme 1CH3-8CH3 NOE are shown in Figures 4-6.

Figure 5: Assignment of heme pyrrole substituents, 5CH3/6CH2, using SHWEFT 1D NOE

experiments. Conditions: truncated BjFixLH(Fe3+) (0.002M) in 95% H2O/0.20M Tris (pH

8.0)/0.10M NaCl. A. 1D absorption spectrum. B. 1D NOE difference spectrum of 5CH3 irradiation. Note NOE to the nearest 6Hn, (n = near).

C. Difference spectrum of 6Hn irradiation, note NOEs to both its geminal partner 6Hf (f = far;

strong NOE) and to the nearby 5CH3 (weak).

D. Difference spectrum of 6Hf irradiation showing strong NOE to its geminal partner, 6Hn.

Figure 6: NOEs detected between 0 and 10 ppm using SHWEFT 1D NOE experiments. Conditions: truncated BjFixLH(Fe3+) (0.002M) in 95% H2O/0.20M Tris (pH 8.0)/0.10M NaCl.

Note the NOE peaks in the difference specta close to the water resonance. A. Difference spectrum from irradiating the 6Hn.

B. Difference spectrum from irradiating the 6Hf.

C. Difference spectrum of heme 2vinyl-H irradiation showing NOEs to the vinyl proton resonances.

Figure 7: Using experiments like those in Figures 5 and 6 it is possible to assign heme methyl/vinyl and methyl/propionate substituents for each heme pyrrole. Specific assignments depend crucially upon making the connection between the heme 1CH3 and 8CH3, shown in this

figure. Conditions: truncated BjFixLH(Fe3+) (0.002M) in 95% H2O/0.20M Tris (pH 8.0)/0.10M NaCl.

A. Absorption spectrum. B. Difference NOE spectrum from irradiation at heme 1CH3.

C. Difference NOE spectrum from irradiation at heme 8CH3.

Making unambiguous complete, stereospecific heme assignments depends upon making at

least one connection between substituents on neighboring pyrrole rings. While intra-pyrrole

connectivities are typically easy, as shown in Figures 4-6, inter-pyrrole connectivities are more

difficult. The reason for this is the comparatively weak NOE magnitudes. Our work on FixLH

depended crucially upon making the connection between the heme 1CH3 and 8CH3, as shown in

Figure 8.

Figure 8: Using experiments like those in Figures 4, 5 and 6 it is possible to assign heme methyl/vinyl and methyl/propionate substituents for each heme pyrrole. Conditions: truncated BjFixLH(Fe3+) (0.002M) in 95% H2O/0.20M Tris (pH 8.0)/0.10M NaCl.

A. Absorption spectrum. B. Difference NOE spectrum from irradiation at heme1CH3.

C. Difference NOE spectrum from irradiation at heme 8CH3.

Figure 9: Heme hyperfine resonance assignments for recombinant truncated Bj FixLH (Fe3+) at 22 °C in 95% H2O/0.20M Tris (pH 8.0)/0.10M NaCl.

1. We have developed several new pulse sequence methods that produce high quality spectra of

high-spin paramagnetic ferriheme proteins, both in D2O and H2O solutions.

2. These methods are quite robust and can be optimized quickly, often by setting just a single parameter (ie the WEFT nulling delay).

3. No post processing manipulations are needed for any of the spectra, in contrast to the simple 1D presat spectra.

4. Receiver gain can be set at 2/3 of maximum for all H2O samples.

5. The seuqences are based on the Super-WEFT and PASE experiments, with the main difference being incorporation of band selective and very wide band shaped pulses, hence genesis of our name “SHWEFT” (SHaped WEFT). 6. We have demonstrated the advantages of these pulse sequences with metMb and subsequently have applied them to making complete, unambiguous assignments of the heme hyperfine resonances of truncated, recombinant BjFixLH.

7. The difference spectra are of high quality, with little baseline distortion and excellent subtractions, both of which allow exceptional identification of NOEs within the diamagnetic envelope, even as

close as within 1 ppm of residual water, in 95% H2O.

8. For the first time complete heme hyperfine resonance assignments have been made for a unique heme oxygen sonsor protein: recombinant truncated BjFixLH (Fe3+; S = 5/2).

1. J.S. de Ropp, G.N. La Mar (1991) J. Am. Chem. Soc. 113, 4348-4354.

2. K. Clark, L. B. Dugad, R. G. Bartsch, M. A. Cuanovich, G. N. La Mar (1996) J. Am. Chem. Soc. 118, 4654-4660.

3. A. Bondon, C. Mouro (1998) J. Magn. Reson. 134, 154-157.

4. MestRe-C: http://gobrue.use.es/jsgroup/MesttRe-C/MestRe-C.html.

5. E. Kupce, R. Freeman (1994) J. Magn. Reson., 108, 268-373.

6. E. Kupce, R. Freeman (1994) J. Magn. Reson., 118, 299-303.

7. M. K. Chan; (2001) Curr. Opinion Struct. Biol. 5, 216-222.

8. M. A. Gilles-Gonzalez, G. Gonzalez, M. F. Perutz (1995) Biochemistry 34, 232-236.

We wish to acknowledge the facilities of the WSU NMR Spectroscopy

Center, supported by NIH(RR0631401; RR12948) & NSF (CHE9115282; DBI9604689)

and the Murdock Charitable trust. JDS wishes to acknowledge support from NIH (GM47645). We

thank Christine Suquet for FixLH sample preparation.