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suggest that the angle formed by the projection of the imidazole ring onto
the porphyrin plane is approximately 60 measured from the N A
N C axis of
reference, pointing towards the 8-CH 3 and the 4-vinyl group. This orien-
tation avoids major steric conflicts between the pyrrole nitrogens and the
C d 2H and C e 1H units of the imidazole ring, presumably allowing greater
freedom of motion to the proximal histidine and the iron ( Samuni, Ouellet,
Guertin, Friedman, & Yeh, 2004 ). The NMR study was performed before
the X-ray structure of a truncated globin became available. In their conclu-
sions, the authors point out that ' ... the folding topology of the compact
globin adheres well to the conventional globin fold'. Although this is not
strictly the case ( Figs. 6.1 and 6.2 ), an overlay of the three-dimensional struc-
tures of CtrHb and sperm whale myoglobin illustrates that the cages formed
around the haem group by the two proteins are strikingly similar. As the first
cyanobacterial TrHb1 to be characterized in some detail, N. commune GlbN
illustrates both the pitfalls of in vitro studies and the power of UV-vis, CD
and NMR spectroscopy as structural tools. No additional structural informa-
tion is available on N. commune GlbN at this time.
5.1.2 C. eugametos LI637 Hb
The optical properties of CtrHb ('H19' construct starting at Ser44), like
those of N. commune GlbN, immediately indicated a behaviour distinct from
that of myoglobin ( Couture & Guertin, 1996 ). The purified protein in the
ferric state has a pH-dependent spectrum showing evidence for a coordi-
nated water at low pH and endogenous coordination at high pH. This sixth
ligand can be displaced by cyanide. The reduced state spectrum resembles
that of N. commune GlbN, also supporting a mixture of low- and high-spin
species. Ferrous CtrHb is able to bind dioxygen reversibly and exhibits a
depressed a / b intensity ratio.
In a follow-up study, Couture et al. (1999) explored further the behav-
iour of the ferrous state. Three distinct protein forms are observed as the pH
is varied: a four-coordinate species at acidic pH, a 5c species with high-spin
character at neutral pH and a low-spin six-coordinate (6c) species at high
pH. This complex response and that of N. commune GlbN support the
extraordinary plasticity of the distal haem pocket. An additional study
resorting to a combination of site-directed mutagenesis, RR and EPR added
to UV-vis absorbance spectroscopy identified the distal ligand to the ferric
iron as Tyr B10. Ionization of the ligating phenol to phenolate for coordi-
nation is presumably aided by a lysine at position E10 ( Das et al., 1999 ).
Replacement of Tyr B10 with a leucine yields an EPR spectrum resembling
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