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Objective: To explore the effect of Heshouwuyin on the expression of cytochrome C oxidase7a2 (Cox7a2) in testis tissue of rats with exercised-induced fatigue., Methods: Fifty SD rats were divided into normal control group (A group), Heshouwuyin administered normal group (B group), model control group (C group), Heshouwuyin treated group (D group) and Heshouwuyin prevented group (E group) randomly with 10 rats for each. The exercise-induced fatigue models in rats of C, D, E groups were established. The rats in D group were treated with Heshouwuyin [20 g/(kg x d), contained crude drug 9.6 g/mL] for 60 days (during the 42 days of modeling and after the 18 days of modeling). The rats in E group were also treated with Heshouwuyin for 60 days (but before the 18 days of modeling and during the 42 days of modeling). Beckmancoulter Unicel Dxl 800 was used to detect the level of serum testosterone, according to the manufacture's instructions. Western blot and RT-PCR were used to observe the differential expression of Cox7a2., Results: The level of serum testosterone in C group was decreased compared with A group (P < 0.05), which implied the success of modeling. Compared with group A, the level of serum testosterone in B, D, E groups were increased (P < 0.05). Cox7a2 protein was expressed mainly in leydig cell and spermatocyte. Compared with A,B, D, E groups, the expression of Cox7a2 protein and mRNA in C group increased (P < 0.05), and there no significant difference was observed between group A and B, as well as group D and E., Conclusion: The expression of Cox7a2 was down-regulated by Heshouwuyin.

Noncovalent bonding interactions of nitric oxide (NO) with human serum albumin (HSA), human hemoglobin A, bovine myoglobin, and bovine cytochrome c oxidase (CcO) have been explored. The anesthetic nitrous oxide (NNO) occupies multiple sites within each protein, but does not bind to heme iron. Infrared (IR) spectra of NNO molecules sequestered within albumin, with NO present, support the binding of NO and NNO to the same sites with comparable affinities. Perturbations of IR spectra of the Cys(34) thiol of HSA indicate NO, NNO, halothane, and chloroform can induce similar changes in protein structure. Experiments evaluating the relative affinities of binding of NO and carbon monoxide (CO) to iron(II) sites of the hemeproteins led to evidence of NO binding to noniron, nonsulfur sites as well. With HbA, IR spectra of cysteine thiols and/or the iron(II) N-O stretching region denote changes in protein structure due to NO, NNO, or CO occupying noniron sites with an order of decreasing affinities of NO > NNO > CO. Loss of NO from some, not all, noniron sites in hemeproteins is very slow (t(1/2) approximately hours). These findings provide examples in which NO and anesthetics alter the structure and properties of protein similarly, and support the hypothesis that some physiological effects of NO (and possibly CO) result from anesthetic-like noncovalent bonding to sites within protein or other tissue components. Such bonding may be involved in mechanisms for control of oxygen transport, mitochondrial respiration, and activation of soluble guanylate cyclase by NO.

Spectroelectrochemical titration studies involving the binding of the infrared-active probe ligand carbon monoxide (CO) to the heme alpha 3/CuB site of bovine heart cytochrome c oxidase (CcO) have been reexamined. The spectroelectrochemical cell employed was constructed to monitor both the infrared (IR) and visible/Soret spectra of the CcO-CO complex as a function of the overall oxidation state of the enzyme. A number of commonly used electron transfer mediators were employed to shuttle electrons between the redox active sites within the enzyme and the electrode surface. The well-documented shift in the CO infrared stretch band maximum from 1963.3 cm-1 (CcO fully reduced) to 1965.5 cm-1 (CcO partially oxidized) was carefully titrated electrochemically. Deconvolution of the asymmetric CO stretches indicates the existence of two different states of CO vibrators within the enzyme, presumably due to two conformers which are present in a ratio of approximately 5:1. Upon incrementally stepping the potential from the fully reduced state to the partially oxidized state, we found it possible to follow the decrease in the intensity of the original pair of these conformers and the concomitant increase of a resultant pair while maintaining this 5:1 ratio between the conformers. By plotting the change in the deconvoluted CO peak intensities vs the redox potential, as well as the absorbance changes in the visible/Soret spectra vs the redox potential, we found not only that both fit an n = 1 electron process but also that the spectral changes tracked each other identically with experimental error. Furthermore, analysis of the second derivative of the Soret spectra allowed for the qualitative monitoring of the oxidation state of the Fe alpha site which again tracked identically to that of the CO shift in the IR region. These results would seem to confirm earlier suggestions that perturbing the oxidation state of Fe alpha causes a conformational change in the enzyme which affects the binding site for CO, namely heme alpha 3. As a consequence of the CO IR stretching frequencies changing by only 2 cm-1 during this redox titration, with no accompanying changes in half band width, we suggest that it is impossible that this small but significant change seen in the CO stretching frequencies could be due to an oxidation state change in CuB, given the known sensitivity of the CO stretching frequency to perturbations and the close proximity of Cu(B) to the CO binding site at heme alpha 3 (4.5 A). Therefore, it would appear that Cu(B) must remain reduced as long as CO is bound to the heme alpha 3 site. This is consistent with earlier proposals that Fe alpha 3 and Cu(B) are acting together as a two-electron donor to dioxygen.

Reduction of nitric oxide (NO) to nitrous oxide (N2O) is catalyzed by bovine heart cytochrome c oxidase (CcO) in anaerobic solutions at pH 7.2 and 20 degrees C. Cyanide inhibits and forms Fea3(3+)CN. The mononitrosyl (Fea3(2+)NO), but not the dinitrosyl (Fea3(2+)NO; CuB+NO), is a likely intermediate in N2O formation. One-electron reduction of NO at Fea3(2+) could yield N2O via HNO. However, a two-electron reduction of the NO ligand to give an intermediate that reacts with a second NO to give N2O and H2O appears more likely. Conversion of NO to N2O is favored by low levels of both NO and O2, higher NO levels can inhibit both cytochrome c oxidase and NO reductase activities. Raising the O2 level will favor catalysis of NO oxidation to NO2 by CcO. The reactions of NO and the specific CcO activity that occur in tissue will be critically dependent on NO, O2, and CcO levels.

Effects of changes in oxidation state at the other metal centers on oxidized heme a3 cyanide of bovine heart cytochrome c oxidase have been investigated. Only one CN- binds, giving Fe3+a3CN, in fully-oxidized cytochrome c oxidase and its 1-, 2-, and 3-electron reduction products. Soret/visible spectra for the heme a3 cyanide are independent of overall redox level, whereas distinct shifts in C-N infrared stretch band frequency occur upon reduction, reflecting changes in the polarity of the ligand (CN-) environment. Catalysis of O2 reduction can be critically dependent upon such changes in polarity at the reduction site. These findings indicate that CuB, when reduced, exists in two forms whose relative stabilities are independent of Fea and CuA oxidation states and, when oxidized, is in only one stable form. These results are consistent with the oxidation of Cu+B triggering proton pumping and with the involvement of a CuB ligand in respiratory control. Electron equivalents introduced into the enzyme are distributed equally among Fea, CuA, and CuB, which raises the possibility that all four electrons used in O2 reduction are donated via Cu+B, which is favorably positioned with respect to Fea3 (the O2 binding site) in order to carry out this role in electron transfers.

Subunit V, one of the nuclear-coded subunits of yeast cytochrome c oxidase, has two isoforms, Va and Vb. These alter the in vivo intramolecular rates of electron transfer within the holoenzyme (Waterland, R. A., Basu, A., Chance, B., and Poyton, R. O. (1991) J. Biol. Chem. 266, 4180-4186). The isozyme with Vb has a higher turnover rate and a higher intramolecular transfer rate than the isozyme with Va. To determine how these isoforms affect catalysis, we have examined their effects on the binuclear reaction center and on the interaction between cytochrome c oxidase and the two isoforms, iso-1 and iso-2, of yeast cytochrome c. Infrared spectroscopy of carbon monoxide liganded to heme a3 has revealed a single conformer for the binuclear reaction center in the isozyme with Vb but two discrete conformers in the isozyme with Va. The kinetics of interaction for all four pairwise combinations of isozymes with each subunit V isoform and the two cytochrome c isoforms are biphasic, with high and low affinity electron transfer reactions. In general, the isoforms of cytochrome c and subunit V do not alter the Km but do affect the TNmax. The TNmax for isozymes carrying Vb are higher at both high and low affinity sites for each cytochrome c isoform. Iso-1-cytochrome c supports a higher TNmax than Iso-2-cytochrome c. Surprisingly, the combinatorial effect of both sets of isoforms on TNmax is minimized with the pairs of isoforms (iso-1-cytochrome c and subunit Va or iso-2 and subunit Vb) that are co-expressed in cells. Together, these findings support the conclusion that the subunit V isoforms modulate catalysis and suggest that they do so by affecting the environment or structure of the binuclear reaction center. They also suggest that the coexpression of the two cytochrome c isoforms with two subunit V isoforms serves to minimize differences in electron transfer rates brought about by the subunit V isoforms.

The stable binding of nitric oxide to both Cu+B and Fe2+a3 in cytochrome c oxidase is shown in infrared spectra. The N-O stretch band for Fe2+a3-NO at 1610 cm-1 is similar to the band for MbNO at 1612 cm-1. The Cu+B-NO band is at 1700 cm-1. Thus, electron donation from metal to NO is greater with Fe2+a3 than Cu+B. However, the affinity for NO is only slightly greater at Fe2+a3 than at Cu+B. In contrast CO binds stably only to Fe2+a3. Infrared spectra of oxidase, myoglobin and hemoglobin NO and CO complexes are consistent with only one stable protein structure at the ligand binding site of nitrosyls and with dynamic multiple protein conformers at carbonyl sites. The binding of NO to both Cu+B and Fe2+a3 of cytochrome c oxidase can contribute to NO cytotoxicity and to the catalysis of NO reduction to N2O and makes possible the effective use of infrared spectroscopy in investigations of Cu as well as Fe at the binuclear O2 reduction sites of oxidases.

IR spectra directly probe specific vibrators in bovine heart cytochrome c oxidase, yielding quantitative as well as qualitative information on structures and reactions at these vibrators. C-O IR spectra reveal that CO binds to Fe2+ a3 as two conformers each in isolated immobile environments sensitive to Fea and/or CuA oxidation state but remarkably insensitive to pH, medium, anesthetics, and other factors that affect activity. C-N IR spectra reveal that the one CN- that binds to fully and partially oxidized enzyme can be in three different structures. These structures vary in relative amounts with redox level, thereby reflecting dynamic electron exchange among Fea, CuA, and CuB with associated changes in protein conformation of likely significance in O2 reduction and H(+)-pumping. Azide IR spectra also reflect redox-dependent long-range effects. The amide I IR bands, due to C-O vibrators of peptide linkages and composed of multiple bands derived from different secondary structures, reveal high levels of alpha-helix (approximately 60%) and subtle changes with redox level and exposure to anesthetics. N2O IR spectra reveal that these anesthetic molecules at clinically relevant levels occupy three sites of different polarity within the enzyme as the enzyme is reversibly, but only partially, inhibited.