The work described in this thesis deals with the
properties, cellular location, effect of growth
conditions and the redox properties of cytochrome c₄
from the bacterium Pseudomonas stutzeri 224.
Spectrally cytochrome c₄ is characterised by an
α-peak maximum at 550nm and an α/ß ratio of 1.22-1.23.
The mobility on SDS gels is dependent on the redox state
of the haem iron and whether the haem is present or not.
The cellular location of cytochrome c⁴ was
identified as being mainly membrane bound by
purification of cytochrome c⁴ from both soluble and
membrane fractions. Analysis of cytoplasmic, periplasmic
and membrane fractions by haem stained gels confirmed
that cytochrome c⁴ is mainly membrane bound and in
addition demonstrated that soluble cytochrome c⁴ is
located in the periplasm. Proteolysis experiments on
right-side-out vesicles demonstrated that cytochrome c^
is associated with the periplasmic face of the membrane.
Cytochromes c⁴ from soluble and membrane fractions of
aerobic and nitrate grown cells were shown to be
identical with respect to amino acid composition,
spectra, ELISA and redox titration.
The levels of cytochrome c⁴ were shown to be
virtually identical in both aerobic and nitrate grown
Pseudomonas stutzeri, however, the distribution of
cytochrome c⁴ between membrane and soluble fractions was
affected by growth conditions with more membrane bound
cytochrome c⁴ found in aerobically grown cells.
Redox titration of cytochrome c⁴ yielded sigmoidal
Nernst plots which may be analysed in terms of two
components of +300 and +190mV. Two models are proposed
to explain the two redox potentials; (1) the haems are
intrinsically different or (2) the haems have identical
potentials in the oxidised form but addition of one
electron makes the addition of a second much more
difficult (negative cooperativity) . Proteolytic cleavage
of the cytochrome with chymotrypsin yielded two
fragments, one with a molecular weight of approx. 10000,
a 695nm band and a midpoint potential of +110mV. The
second fragment was damaged, as determined by the loss
of the 695nm band, had a molecular weight of approx.
6000 and a midpoint potential of -190mV. Without two
undamaged fragments it was not possible to distinguish
between the two proposed models of reduction.
A partial characterisation of a c-type cytochrome
of approx. 30000 molecular weight, which is greatly
induced under nitrate growth, is also reported.