NUCLEAR MAGNETIC RESONANCE 117
have been found to be in excellent agreement with experimentally determined
values in solution. Methods that improve structure refi nement in NMR solu-
tion studies include the use of a conformational database potential and direct
refi nement against three - bond coupling constants (^3 J values), secondary^13 C
shifts,^1 H shifts, T 1 / T 2 ratios, and residual dipolar couplings. Residual dipolar
couplings (RDCs), yield long - distance restraints not accessible by other solu-
tion NMR parameters. The article by Clore and Gronenborn gives an excellent
overview of the new refi nement strategies that increase the accuracy of
solution NMR structures.^23
3.4.12 Descriptive Examples,
Cytochromes c (see Section 7.7 ) are ubiquitous in nature. This electron trans-
fer protein is relatively small ( ∼ 12.5 kDa, ∼ 100+ amino acid residues) and fea-
tures high solubility, high helical content, thermodynamic stability, and a
spectroscopically accessible iron - containing heme cofactor. In eukaryotes,
cytochrome c mediates single electron transfer between mitochondrial inner
membrane enzymes cytochrome bc 1 (Section 7.6 ) and cytochrome c oxidase
(Section 7.8 ). Cytochromes bc 1 and cytochrome c oxidase are known as com-
plexes III and IV, respectively, of the physiological respiratory chain. Cyto-
chromes c are characterized by the attachment of the heme c cofactor to the
protein chain through thioether linkages provided by two cysteinyl residues
covalently bonded to vinyl substituents of the heme porphyrin ligand. (See
Figures 7.25 and 7.32 .) In addition to the four porphyrin nitrogen ligands,
cytochrome c ’ s heme iron ion carries one axial histidine ligand. Some variety
exists for the iron ion ’ s second axial ligand, although it is often the S δ atom of
a methionine residue.
Recently, Sivakolundu and Mabrouk published an NMR solution structure
of horse heart ferrocytochrome c in which the heme contains an Fe(II) ion.^24
This solution structure was the fi rst in which the cytochrome c protein was
dissolved in a nonaqueous solvent: a solvent mix of 70% water and 30% ace-
tonitrile (ACN). The data obtained from the NMR study are deposited in the
protein data bank (PDB) as: (1) 1LC1, the minimized average NMR structure;
and (2) 1LC2, the 30 lowest energy NMR structures.
The ferrocytochrome c features a 104 - amino - acid residue single chain with
secondary structure consisting of fi ve α - helices, two omega loops, and several
random coil segments. The c - type heme is attached covalently to the polypep-
tide chain through cys14 and cys17. The signature CXXCH sequence found in
cytochromes c exists in this ferrocytochrome as cys14 – ala15 – gln16 – cys17 –
his18. His18 and met80 form the heme Fe(II) ’ s axial ligands. His 18 is bonded
through its N ε 2 side - chain atom with a N ε 2 – Fe = 1.97 Å bond length. Met80 is
bonded through its S δ atom with S δ – Fe = 2.68 Å , an atypically long Fe – S
bond length. Much more detail on this and other cytochromes c is found in
Section 7.7. Here we will discuss how the NMR structure was obtained
experimentally.
