CAPÍTULO II MARCO TEÓRICO
MUNICIPALIDAD DISTRITAL DE PILLCO MARCA
Carbon monoxide binding to cytochrome o was confirmed by the difference spectrum in the Soret region showing a peak at 415nm and a trough at 432nm, in agreement with reported results in the literature (Caster and Chance, 1955,1959). This has already been reported in Chapter 3. Carbon monoxide difference spectra (reduced-pius-CO minus
Potentiometry of cytochrome o
reduced) in the region of SOOnm to 600nm showed two additional peaks at 530nm and 568nm and troughs at 550nm and 580nm (see Figure 3.5b).
Figure 4.3 shows the difference spectra obtained from pur i f i ed cytochrome o between 600nm and 800nm in the presence and absence of carbon monoxide. The reduced minus
oxidised difference spectrum reveals a feature centred around 758nm (Figure 4.3, upper trace) . The ratio of the Soret/758nm bands is approximately 1000, which would make the extinction coefficient of this feature about 0.145 mM"lcm"l assumming 1 0 0% of the haem contributes to this feature (calculated from the G^Kita et ai., 1984a) . The 758nm band is quenched by the addition of carbon monoxide (Figure 4.3, lower trace) and subsequently regenerated by prolonged aggitation in the presence of dithionite and absence of carbon monoxide. The carbon monoxide bleaching can also be reversed by light, in parallel to other carbon monoxide binding characteristics, (not sh own ). Membrane
fragments obtained from strain RG145 also show a peak at 758nm which confirms this feature is real and not an artifact of the purification procedure. The absorbance associated with the membrane bound form of the enzyme is also quenched by carbon monoxide as well as other inhibitory ligands known to bind to the reduced form of high-spin haem
(not shown).
The optical spectra of myoglobin and haemoglobin have been extensively studied with the result that the optical bands of these proteins have been well characterised.
The effect of carbon monoxide on the 758nm absorbance. , Spectra of purified cytochrome o, reduced with solid dithionite, were scanned in the near infrared region from 700nm to 900nm, in the presence and absence of carbon monoxide. Loss of the 758nm band is seen in the carbon monoxide-inhibited oxidase (lower trace). Spectra were recorded at room temperature, in 50mM phosphate buffer, pH7.5, the protein concentration was 3|IM, final.
AA
0.001
CO
+ CO
700 800 900
Absorbance spectra in the near infrared region of myoglobin, Mb(Fe2+), show an absorbance peak at 758nm (lizuka et aJ., 1974) which is sensitive to the conformational state of the haemoprotein (Xie & Simon, 1991; Ansari et ai., 1985; Campbell et al., 1987; Sassaroli & Rousseau, 1987; Flamingo
St Alben, 1985; Chavez et ai., 1990) . This absorbance arises from a charge transfer transition between the porphyrin % system and the iron K system > d%y] (Eaton et
ai., 1978) . The Mb (Fe^**") ~C0 form is also reported to have an absorbance in this region but this broadens markedly on increasing the temperature (almost negligible above SDK). In this respect, we would expect to see a near infrared a b s o r b a n c e f e a t u r e d i s a p p e a r in t h e c a r b o n monoxide-inhibited cytochrome o at room temperature. This is what is observed (see Figure 4.3, iower trace). Photolysis of the Mb(Fe2 + )-C0 species gives rise to the reduced, unliganded, species. However, at s u f f i c i e n t l y low temperature (4.2K) the 758nm band of myoglobin is shifted to 772nm signifying a conformational difference between the reduced form (R, ground state) and the reduced form created by photodissociation (R^) (lizuka et a l ., 1974). No red shift has so far been observed in the photodissociated cytochrome o enzyme. This is possibly because at 77K, the lowest temperature at which photolysis experiments have so far been attempted (not shown), the photolysed enzyme can relax to the reduced ground state. Lower temperature (<50K) photolysis experiments are required to identify the cytochrome o enzyme in the ligand bound conformation but in
Potentiometry of cytochrome o
the non-liganded state (R*).
Future studies might also benefit from coupling mutagenesis experiments on putative endogenous ligands to the high-spin haem o with optical studies in the near infrared region.
Carbon monoxide also causes a red shift in the high-spin trough at 632nm, this is shown in Figure 4.4. In the presence of carbon monoxide the trough (inverted by computer manipulation and shown as a peak in figure) broadens with a shift in the to 643nm (lower trace). Since the 632nm absorbance is thought to be due to a ferric
(Fe3+) haem species it is not expected to be present in the c a r b o n m o n o x i d e - i n h i b i t e d en zy m e ( F e ^ ^ - C O ) . The "corresponding" high-spin ferric haem absorbance in cytochrome c oxidase (655nm) is abolished by carbon monoxide.
4,2.4 Redox behaviour of the (X band
Optical titrations were performed at pH 6, 7 and 8 and the two peaks in the a band (X-^ax 556nm and 564nm) associated with cytochrome o were deconvoluted using 57 6nm as the reference wavelength. The titration curves for each peak can be seen in Figure 4.5a. Each curve contained two separable components which fit well with two n=l processes. Each transitio n showed ap proximately - 6 0 m V/pH unit dependence over the pH range, (Figure 4,5b). At pH 7.0 the feature centred at 564nm gave E#,? values of +24 9mV for the
F,i.gure 4^4.
The effect of carbon monoxide on the high-spin trough. ,
Shown are difference spectra of cytochrome o in the 600nm and 700nm optical region. For the purposes of this diagram, the trough has been inverted by subtracting a fully reduced spectrum from a sample spectrum. The upper trace is of the enzyme in the absence of carbon monoxide and is essentially an oxidised minus reduced spectrum. It can be seen that an oxidised (ferric) absorption feature is centred at 632nm. The lower trace was produced by treating the sample with carbon monoxide and represents the carbon monoxide-inhibited spectrum minus the fully reduced. The lower trace shows the trough shifted ~10nm (^max 643nm) towards the red and broadened by carbon monoxide. Conditions were the same as those for Figure 4.3, except, final protein concentration was 0.9jIM.