Backbone 1H, 13C and 15N Chemical Shift Assignments for Reduced Human Ferredoxin

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Xia, B., Volkman, B. F. , and Markley, J. L., "Evidence for Redidation-State-Dependent
Conformational Changes in Human Ferredoxin from Multinuclear, Multidimensional NMR
Spectroscopy," Biochemistry 37, 3965-3973 (1998).

Evidence for oxidation-state-dependent conformational changes in human ferredoxin from multinuclear, multidimensional NMR spectroscopy.

Human mitochondrial ferredoxin is a [2Fe-2S] protein that functions to transfer
electrons from NADPH-dependent ferredoxin reductase to cytochrome P450 enzymes.
Two of the three histidines of human ferredoxin are strictly conserved in the
sequences of all known vertebrate ferredoxins, and one of these (His56) is
adjacent to Cys55, which serves as one of the ligands to the iron-sulfur
cluster. All but 16 of its residues show sequence identity with those of bovine
ferredoxin. It has been proposed for bovine ferredoxin that His56 hydrogen
bonds with a labile sulfur and that the reduction of the iron-sulfur center is
accompanied by the uptake of a proton by this histidine [Lambeth, J. D.,
Seybert, D. W., Lancaster, J. R., Jr., Salerno, J. C., & Kamin, H. (1982) Mol.
Cell. Biochem. 45, 13-31]. In this paper, we report procedures for labeling
human ferredoxin uniformly with 15N using 15NH4Cl and selectively with 13C by
the incorporation of [U-13C]histidine. Most of the imidazole 1H, 13C, and 15N
resonances of the three histidines have been assigned by heteronuclear
two-dimensional single- and multiple-bond correlation spectroscopy.
Site-directed mutagenesis was used in assigning the NMR signals from His56. The
pKa values of His10 (6.5) and His62 (5.8) in oxidized human ferredoxin were
found to be similar to those reported previously for the corresponding residues
of bovine ferredoxin [Greenfield, N. J., Wu, X., & Jordan, F. (1989) Biochim.
Biophys. Acta 995, 246-254; Miura, S., Tamita, S., & Ichikawa, Y. (1991) J.
Biol. Chem. 266, 19212-19216].(ABSTRACT TRUNCATED AT 250 WORDS)

Xia, B., Cheng, H., Skjeldal, L., Coghlan, V. M., Vickery, L. E.,
and Markley, J. L., Biochemistry 34, 180-187, (1995).

Multinuclear magnetic resonance and mutagenesis studies of the histidine residues of human mitochondrial ferredoxin.

Human ferredoxin, the human equivalent of bovine adrenodoxin, is a small
iron-sulfur protein with one [2Fe-2S] cluster. It functions, as do other
vertebrate ferredoxins, to transfer electrons during the processes of steroid
hormone synthesis. A DNA fragment encoding the mature form of human ferredoxin
was cloned into an expression vector under control of the T7 RNA
polymerase/promoter system. The protein was overproduced in Escherichia coli,
and the [2Fe-2S] cluster was incorporated into the protein by in vitro
reconstitution. The overall yield was approximately 30 mg of purified,
reconstituted ferredoxin per liter of culture. Four of the five cysteines in
human ferredoxin are coordinated to the iron-sulfur cluster. First, the
non-ligand cysteine (cysteine-95) was mutated to alanine, and then double
mutants were created in which each of the other four cysteines (at positions
46, 52, 55, and 92) were mutated individually to serine. The wild-type
ferredoxin and each of the five mutant proteins were studied by UV-visible
spectroscopy and electron paramagnetic resonance spectroscopy. The EPR gav
values of all five mutants were very similar to that of wild-type human
ferredoxin. In the reduced state, three of the cysteine-to-serine mutants
exhibited axial EPR spectra similar to that of wild-type, but one of the double
mutants (C52S/C95A) exhibited a rhombic EPR spectrum. The UV-visible
spectroscopic properties of the wild-type and the C95A mutant ferredoxins were
identical, but those of the other cysteine-to-serine mutant proteins of human
ferredoxin were quite different from those of the wild-type protein and each
other. These results, along with those from cysteine-to-serine mutations in
other ferredoxins, provide the basis for a more comprehensive theoretical and
practical understanding of the features important to the ligation of [2Fe-2S]
clusters, although they do not yet permit determination of which two cysteines
ligate Fe(II) and which ligate Fe(III) in the reduced protein.

Xia B., Cheng H., Bandarian V., Reed G. H., & Markley J. L.,
Biochemistry 35, 9488-9495,(1996).

Human ferredoxin: overproduction in Escherichia coli, reconstitution in vitro, and spectroscopic studies of iron-sulfur cluster ligand cysteine-to-serine mutants.

Chylla, R. A. & Markley, J. L. J. Magn. Reson. (Series B) 102, 148-154, (1993)

Crystal Structure Of Human Cyp11a1 In Complex With Cholesterol

Crystal Structure Of Human Cyp11a1 In Complex With 22- Hydroxycholesterol

Crystal Structure Of Human Cyp11a1 In Complex With 20,22- Dihydroxycholesterol

Crystal Structure Of Human Cyp11a1 In Complex With 20- Hydroxycholesterol

Crystal Structure Of Human Ferredoxin-1 (Fdx1) In Complex With Iron- Sulfur Cluster

adrenodoxin, mitochondrial precursor [Homo sapiens]

PREDICTED: hypothetical protein LOC709110 [Macaca mulatta]

PREDICTED: adrenodoxin, mitochondrial [Nomascus leucogenys]

PREDICTED: adrenodoxin, mitochondrial [Papio anubis]

PREDICTED: adrenodoxin, mitochondrial, partial [Saimiri boliviensis boliviensis]

RecName: Full=Adrenodoxin, mitochondrial; AltName: Full=Adrenal ferredoxin; AltName: Full=Ferredoxin-1; AltName: Full=Hepatored

SUMMARY TABLE: Spectral Parameters for the NMR Data Sets Collected on Human Ferredoxin
d1 (1H)b d2 d3 Matrix Size
Experiment SF(/MHz) SW (/Hz) Nucleus SW (/Hz) N2c Nucleus SW(/Hz) N3c (d1 x d2 x d3)
2D 1H-15N HSQC 500.13 6009.615 15N 1666.67 256 512 x 25
3D HNCO 500.13 6009.615 13C' 2000.00 64 15N 1666.67 36 512 x 256 x 128
3D HNCA 500.13 6009.615 13Ca 4545.45 60 15N 1666.67 32 512 x 256 x 128
3D HN(CO)CA 500.13 6009.615 13Ca 4545.45 64 15N 1666.67 28 512 x 256 x 128
3D HNCACB 500.13 6009.615 13Ca/b 7352.94 64 15N 1666.67 32 512 x 256 x 128
3D CBCA(CO)NH 500.13 6009.615 13Ca/b 7352.94 42 15N 1666.67 32 512 x 256 x 128
3D HCACO 500.13 6009.615 13C' 2000.00 64 13Ca 4545.45 32 512 x 256 x 128
3D 15N-TOCSYd 750.13 8992.806 1H 8992.806 128 15N 2500 32 512 x 512 x 128
3D 15N-NOESYd 750.13 8992.806 1H 8992.806 128 15N 2500 32 512 x 512 x 128
3D 15N-TOCSYe 600.13 6944.444 1H 6944.444 150 15N 1818.18 40 512 x 512 x 128
3D 15N-NOESY 600.13 6944.444 1H 6944.444 150 15N 1818.18 40 512 x 512 x 128
*Summary Table was provided by the author.
a-The parameters listed here apply to NMR data sets for both redidized and reduced human Ferredoxin, except that
3D 15N-TOCSY and 3D 15N-NOESY.
b-A total of 1024 complex points were collected in the 1H dimension (d1) of all experiments. In all experiments
except HCACO, the right half of the spectrum (upfield of H2O) in d1 dimension contained no signals and was discarded.
c-N2 and N3 are the numbers of complex points collected for d2 and d3 dimensions, respectively.
d-NMR experimental data collected for redidized HuFd.
e-NMR experimental data collected for reduced HuFd.