CYTOCHROME bc 1 : A BACTERIAL CYTOCHROME 405
structures are the best resolved of any discussed here so far (PDB: 3BCC,
resolution 3.70 Å versus PDB: 1NTK, resolution 2.60 Å ). The inhibitor
antimycin A was thought to bind near heme b H before X - ray crystallography
confi rmed this fact for two reasons: (1) The heme b H UV – visible spectrum
shifted to longer wavelengths (red shift) in the presence of antimycin A, and
(2) the fl uorescence of antimycin A was quenched when it was added to the
cytochrome bc 1 complex.^91 The observed EPR signal of the semiquinone
(QH − • ) is eliminated by antimycin A, consistent with predictions of the Q cycle
that a Q i site inhibitor will inhibit the transfer of electrons from heme b H to
ubiquinone (Q) or ubisemiquinone (QH − • ).^92 It has also been postulated that
conformational change in several subunits of the bc 1 complex would couple
oxidation – reduction events taking place at both the Q o and Q i sites. For
instance, different positions of the ISP protein detected by X - ray crystallo-
graphic studies (as discussed in Sections 7.6.2 and 7.6.3) might indicate a link
between the bifurcation of electron transfer from ubiquinol to cytochrome b
at the Q o site and proton - pumping at the Q i site where antimycin A binds.^93
These predicted conformational changes involving both the Q o and Q i sites
with and without inhibitors have not been detected in X - ray crystallographic
studies (see Table 7.6 , for instance). These include those discussed above. Spe-
cifi cally, the PDB: 1NTZ structure with the substrate substitute ubiquinone - 2
in both sites has virtually the same overall conformational structure as PDB:
1NTK with antimycin A in the Q i site. A conformation comparison between
the PDB: 1PP9 structure (stigmatellin in the Q o site and ubiquinone - 2 in the
Qi site) and PDB: 1PPJ (stigmatellin in the Q o site and antimycin A in the Q i
site) by Berry and co - workers also indicates little conformational change upon
antimycin A binding.^90 (See Figure 7.30 .)
The Berry group researchers provide a detailed analysis of antimycin A
binding in the Q i site. They identify six well - resolved water molecules that
connect antimycin A through a network of hydrogen bonds to aa residues
ser28, asn32, ser35, his201, ser205, lys227, asp228, and others. Their data are
collected in Tables 4 and 6 of reference 90. Of this group, his201 and ser205
are believed to be most important in binding substrate to the Q i site so that
inhibitor binding at these residues could prohibit binding of substrate mole-
cules. Many close contacts between antimycin A and heme b H can explain
antimycin ’ s fl uorescence quenching and UV – visible spectral shifts for heme
bH upon antimycin A binding. There are Ar – Ar contacts between antimycin
A ’ s formylaminosalicylate headgroup and phe220 and between the dilactone ’ s
methyl group and methyl groups on heme b H ’ s pyrrole rings A and B. The
following data use the antimycin A numbering convention of PDB: 1PPJ (ref-
erence 90 ), which, unfortunately, differs from that used for the PDB: 1NTK
structure. The two numbering systems are shown in Figure 7.29. Interactions
with ser35, his201, lys227, and asp228 are as follows: (1) The hydroxyl group
of the formylaminosalicylate ring interacts with the O γ of ser35 (O 1 · · · H 2 O =
2.9, H 2 O · · · S35 O γ = 3.0); (2) the carbonyl oxygen of the amide linker interacts
with the N ε 2 atom of his201 through an intervening water molecule (O 3 · · · H 2 O