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Unusual cytochrome a₅₉₂ with low PO₂ affinity correlates as putative oxygen sensor with rat carotid body chemoreceptor discharge¹

Institut für Physiologie der Universität Rostock,18057 Rostock, Germany; and
^* Max Planck Institut für Molekulare Physiologie, 44227 Dortmund, Germany

²Correspondence: Gertrudenstrasse 9, 18057 Rostock, Germany. E-mail: tino.streller{at}medizin.uni-rostock.de

SPECIFIC AIMS

Oxygen-sensitive processes triggering ion channel conductivity or gene expression are assumed to be initiated by mitochondrial and nonmitochondrial oxygen-sensing cytochromes within the carotid body (CB) located at the carotid artery bifurcation-sensing arterial blood PO₂ changes. We used experimental and mathematical deconvolution of CB light absorption spectra to examine mitochondrial cytochrome c oxidase (CCO) characteristics in order to specify the electron pathway within CCO linked to an unusual low O₂, CO, and high cyanide (CN^-) affinity of CB chemoreceptor nervous discharge (CND).

PRINCIPAL FINDINGS

1. Spectral CB responses
Excised rat carotid bodies were exposed in vitro to a PO₂ of 100 mm Hg (n=5), 80 mm Hg (n=7), 60 mm Hg (n=8), and 0 mmHg (n=4) as well as to cyanide concentrations of 10 µM (n=8), 20 µM (n=9), 30 µM (n=8), 50 µM (n=7), and 100 µM (n=8) in the saline superfusion medium. Figure 1 A, B shows examples for hypoxia and Fig. 1C, D for CN^- -induced light absorption spectra. Spectral deconvolution between 510 and 630 nm revealed high contribution of mitochondrial cytochrome a3₆₀₃, c₅₅₀ and b₅₆₃ and a lower but significant contribution of nonmitochondrial cytochrome b₅₅₈ and of cytochrome a₅₉₂ (black area in Fig. 1A, C ). The relative spectral weight of cytochrome a₅₉₂ decreased significantly (P<0.001) with decreasing PO₂ levels as well as increasing CN^-. In contrast, spectral weights of the other four cytochromes show no significant dependence on PO₂ or CN^- changes.

View larger version (47K): [in this window] [in a new window]   Figure 1. Hypoxia (A, B) and CN- (C, D) -induced light absorbance redox difference spectra of the rat CB are shown as original tracings (noisy curve) and fitted (thick curve) by five different cytochromes: cytochrome c550 (diagonal upward lines), cytochrome b558 (gray shaded), cytochrome b563 (diagonal downward lines), cytochrome a592 (dark shaded), and cytochrome a603 (vertical lines). Spectra under mild hypoxia and low cyanide require a significant spectral weight of cytochrome a592 for fitting.

2. Afferent CND responses
Stimulation of CB tissue by hypoxia or CN^- induced an increase in afferent CND activity. Generally, the time course of the afferent signal during stimulation was dominated by a first transient phasic component, which then became a rather stable and constant tonic component. The time course of CND afferent signal was analyzed for PO₂ levels of 100 mm Hg (n=8), 80 mm Hg (n=7), and 60 mm Hg (n=6). The phasic component of nerve reaction dominated more and more with increasing levels of hypoxia whereas the contribution of the tonic component to chemoreceptor discharge decreased significantly (P<0.01). With increasing CN^-, tonic weight decreased significantly (P<0.01) also.

3. Cytochrome a₅₉₂ and CND response
A highly significant correlation (P>0.01) could be seen between cytochrome a₅₉₂ spectral weight and chemoreceptor discharge under different PO₂ and CN^- values as shown in Fig. 2 A, B. Spectral weight of cytochrome a₅₉₂ dominates at high PO₂ or low CN^- and diminishes with decreasing PO₂ and increasing CN^-, whereas the tonic discharge component is gradually replaced by phasic peak discharge.

View larger version (13K): [in this window] [in a new window]   Figure 2. Tonic weight of chemoreceptor discharge signal vs. spectral weight of cytochrome a592 at different levels of hypoxia (A) and CN- (B) in the medium superfusing CB.

Combined CCO ligand stimulation
Light induced a photolytic inhibition of chemoreceptor discharge stimulated by CO (PCO=500 mmHg) under normoxia (PO₂=200 mmHg). CO binds to CB CCO cytochrome a3 without detectable cytochrome a₅₉₂ spectral weight in the simultaneously recorded spectrum. The photolytic effect on CO stimulated chemoreceptor discharge was inhibited or even inversed at a PO₂ of 60 mm Hg (n=6) as well as at 30 µM CN^- (n=4) in the superfusion medium most likely due to an inhibition of the light-induced photolytic release of CO cytochrome a3 binding at severe reduction of Cu_B. The cytochrome a₅₉₂ spectral weight measurable under these reducing conditions amounted to 4.3% under hypoxia-CO and to 2.7% under CN^--CO stimulation. Both spectral weight values are significantly smaller than under hypoxia or CN^- alone (P<0.01).

4. Spectral responses of cervical and nodose ganglion
Comparative light absorption measurements were carried out on cervical and nodose ganglia (n=8). Similar to CB tissue, light absorption spectra of ganglion tissue showed two distinct peaks during exposure to 3 min hypoxia (PO₂=80 mm Hg). The peaks could be fitted by cytochromes a3₆₀₃, c₅₅₀, b₅₅₈, and b₅₆₃. Cytochrome a₅₉₂ was not detectable. Exposure to high cyanide concentration (100 µM) evoked stable absorption spectra composed mainly of cytochromes a3₆₀₃ and c₅₅₀.

CONCLUSIONS AND SIGNIFICANCE

CCO characteristics
This study describes the relationship between cytochrome redox state as measured by light absorption spectra and carotid body chemoreceptor discharge under different levels of O₂, CN^-, and CO. By means of mitochondrial cytochrome c₅₅₀, b₅₆₃, a₅₉₂, a3₆₀₃, and nonmitochondrial cytochrome b₅₅₈ we were able to deconvolute and fit the experimental spectra with high fidelity. The change in optical density of difference spectra is related to the changed redox state of cytochromes vs. the control state as well as to the number of reducible molecules of each cytochrome. Consequently, spectral weights of cytochromes with low control reduction state increase approximately proportional to increasing reducing conditions, leading to constant spectral weight values. In contrast, a cytochrome already reduced at control will be less reduced in relation to the other cytochromes at increasing reducing conditions, with a subsequent decrease in relative spectral weight. Our results show that the relative weight of cytochrome a₅₉₂ obtained under mild hypoxic conditions (PO₂=100 mmHg) is high with 15.4% and decreases with more severe hypoxia, reaching negligible values under anoxia. Results suggest cytochrome a₅₉₂ is particularly sensitive to changes at higher PO₂ range. When assuming cytochrome a₅₉₂ as the cyanide insensitive cytochrome a component of CB CCO, the normal absorption maximum of cytochrome a at 605 nm is blue shifted to 592 nm. A similar shift for cytochrome a is described in Paracoccus denitrificans as a result of a single site mutation of Arginin 45 hydrogen bonded to the formyl group of cytochrome a to methionine. The relative contribution of cytochrome a and a3 in different spectra were visible, which normally are not separated due to spectral overlap. A drastically lowered midpoint potential of cytochrome a was measured in the mutant enzyme and, correspondingly, a dominance of direct Cu_A to cytochrome a3-Cu_B electron transfer over the normal electron pathway from Cu_A-cytochrome a to cytochrome a3-Cu_B.

Cytochrome a₅₉₂ as oxygen sensor
The dominance of direct electron transfer from Cu_A to cytochrome a3-Cu_B bypassing cytochrome a might suggest cytochrome a₅₉₂ as a cytochrome with an apparent low PO₂ affinity, contrasting the high PO₂ affinity of regular CCO. This result fits the hypothesis of a low PO₂ affinity cytochrome in CB tissue. With cytochrome a as the side of proton pumping and the mitochondrial transmembrane potential (MTP) setting, the unusual low PO₂ affinity of cytochrome a₅₉₂ might be linked to MTP changes in glomus cells over a physiological range of PO₂ values. We may consider cytochrome a₅₉₂ a unique oxygen sensor in carotid body tissue. This assumption is strongly supported by the redox spectra recorded from cervical and nodose ganglion tissue and oxygen-sensing HepG2 cells. Here, the relative contribution of cytochrome c₅₅₀, b₅₆₃, b₅₅₈, and a3₆₀₃ to the spectra is comparable to CB tissue, whereas the unusual cytochrome a₅₉₂ is not detectable. Depolarization of MTP at PO₂ values close to zero in dissociated chromaffin cells and sensory neurons agrees with our spectral analysis of a normal CCO with a high PO₂ affinity in these tissues.

6. CCO ligand interaction
The relative spectral weight of cytochrome a₅₉₂ decreases with increasing degree of hypoxia and increasing CN^- concentration whereas the weights of the other cytochromes remain constant. Thus, cytochrome a₅₉₂ already being reduced under control conditions seems to become more reduced at low CN^- and mild hypoxia indirectly caused by high-affinity ligand binding to the cytochrome a3-Cu_B center and changed electron transfer conditions within the unusual CB CCO. This mechanism induces under CN^- and hypoxic conditions a highly significant correlation between cytochrome a₅₉₂ spectral weight and chemoreceptor discharge tonic weight (Fig. 2) . CO binding to the cytochrome a3-Cu_B center induces under normoxia a tonic chemoreceptor discharge that is inhibited by light due to photolysis of the CO cytochrome a3 binding. Inhibiting photolysis with hypoxia or CN^--induced severe Cu_B reduction as indicated by the abolished or even inversed light effect on chemoreceptor discharge. Tonic or phasic discharge weight values obtained under these conditions might be used to estimate the spectral weight values for cytochrome a₅₉₂ from dose response curves in Fig. 2 . The spectral weight of cytochrome a₅₉₂ under normoxic CO stimulation can be extrapolated. According to Fig. 2A , a tonic discharge response of nearly 100% as seen under normoxic CO stimulation corresponds to a slight hypoxic state or a spectral weight of cytochrome a₅₉₂ of 20%.

Hypothesis
The nonlinear redox changes of cytochrome a₅₉₂ probably induced by O₂, CN^-, and CO binding to the cytochrome a3-Cu_B center of CB CCO can be easily be related the hyperbolic PO₂ vs. chemoreceptor discharge response curve as shown in Fig. 3 . It is conceivable that cytochrome a₅₉₂ mediates the first steps of transmitter release and chemoreceptor discharge. This putative process is likely to be completed by other oxygen sensor cytochromes within the carotid body tissue.

View larger version (16K): [in this window] [in a new window]   Figure 3. Two-cytochrome model of CB oxygen-sensing: CCO mediates electron transfer from the outside to the inside of the inner mitochondrial membrane coupled with proton pumping from the inside to the outside of the membrane. Electrons are supplied by cytochrome c and are normally carried over to cytochrome a3-CuB via CuA and cytochrome a. With cytochrome a592, the bulk flow of electrons is transferred directly to the binuclear center. Part of the protons (HC+) is consumed with the dioxygen reaction, the other part (HP+) for proton pumping generating MTP. Due to electron bypassing, cytochrome a592 dominates with an apparent low PO2; CN- affinity triggers MTP changes and intracellular calcium levels in a nonlinear fashion. In concert with cytochrome a592, an unidentified isoform of the NAD(P) oxidase enhances by closing potassium channels under hypoxia intracellular calcium levels leading to transmitter release and nervous spike generation. The final outcome is represented by the nonlinear PO2–nervous discharge relationship.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0166fje; to cite this article, use FASEB J. (June 21, 2002) 10.1096/fj.02-0166fje

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