Cytochrome oxidases reaction mechanisms

By the procedure described in the foregoing section, cytochrome a is purified and no other cytochrome components are observed spectro-photometrically, so that the sample is sufficiently pure to investigate the mechanism of the cytochrome oxidase reaction. However, there is sometimes a small absorption shoulder at about 430 m/A. To remove... 

Chemical structure and reaction mechanisms of cytochrome c oxidase. R. Lemberg, Rev. Pure Appl. Chem., 1965,15,125-136 (132). 

Cytochrome P450 is considered the most versatile biocatalyst known. The actual reaction mechanism is complex and has been briefly described previously (Figure 11-6). It has been shown by the use of that one atom of oxygen enters R—OH and one atom enters water. This dual fate of the oxygen accounts for the former naming of monooxygenases as mixed-function oxidases. The reaction catalyzed by cytochrome P450 can also be represented as follows ... 

Adults require 1-2 mg of copper per day, and eliminate excess copper in bile and feces. Most plasma copper is present in ceruloplasmin. In Wilson s disease, the diminished availability of ceruloplasmin interferes with the function of enzymes that rely on ceruloplasmin as a copper donor (e.g. cytochrome oxidase, tyrosinase and superoxide dismutase). In addition, loss of copper-binding capacity in the serum leads to copper deposition in liver, brain and other organs, resulting in tissue damage. The mechanisms of toxicity are not fully understood, but may involve the formation of hydroxyl radicals via the Fenton reaction, which, in turn initiates a cascade of cellular cytotoxic events, including mitochondrial dysfunction, lipid peroxidation, disruption of calcium ion homeostasis, and cell death. 

In all protein-protein complexes studied to date in which cytochrome c has been a partner, it has been shown that the ET rates depend strongly on the reaction driving force. It follows that variations in the reorganization energy could control ET rates in these cases [12]. In redox enzymes with two or more active centers, ET between two centers could be turned on by lowering X at roughly constant — AG [1]. Indeed, a proposal has been advanced that this type of mechanism would be an efficient way to gate the electron flow in a redox-linked proton pump such as cytochrome oxidase [75]. 

Irradiation of a CO-adduct of cytochrome oxidase induces the transfer of a CO molecule, originally bound to an iron(II) center, to copper(I) within 1 ps [108]. Photoinitiated dissociation of CO actually occurs in less than 100 fs, probably on the time scale comparable with a vibrational period of the Fe-CO stretch ( 64 fs). Since the involved reaction centers, Fe(II) and Cu(I), are very close, the mechanism in which the Cu-CO bond begins to form as the Fe-CO bond breaks, seems to be a plausible description of the CO transfer. A similar mechanism is believed to hold for the 02 transfer in biological processes in which cytochrome oxidase participates. 

Copper is an essential element to most life forms. In humans it is the third most abundant trace element only iron and zinc are present in higher quantity. Utilization of copper usually involves a protein active site which catalyzes a critical oxidation reaction, e.g., cytochrome oxidase, amine oxidases, superoxide dismutase, ferroxidases, dopamine-/ -hydrox-ylase, and tyrosinase. Accordingly, animals exhibit unique homeostatic mechanisms for the absorption, distribution, utilization, and excretion of copper (J). Moreover, at least two potentially lethal inherited diseases of copper metabolism are known Wilson s Disease and Menkes s Kinky Hair Syndrome (I). 

Haem proteins that react with oxygen also utilise ferryl intermediates. Fig. 4 compares the (proposed) reaction mechanisms of cytochrome oxidase and cytochrome P-450 with those of peroxidases and catalases. As can be seen, several of the reaction intermediates have the same oxidation states (although the protonation steps and stage at which H2O is released may be different). However, in contrast to peroxidases, oxidases must react with molecular oxygen, and this requires a reaction cycle that includes Fe11. 

Fig. 4. A comparison of peroxidase, cytochrome c oxidase and cytochrome P-450 reaction mechanisms. Peroxidase mechanism adapted from that of Poulos[143], cytochrome P-450 mechanism adapted from that of Sligar [100], and cytochrome oxidase mechanism adapted from that of Babcock and Wikstrom[90]. RH, organic substrate for peroxidase and cytochrome P-450 (in the latter case the substrate is presumed to remain bound to the enzyme through most of the reaction cycle).

In order to elucidate the reaction mechanism of cytochrome c oxidase, the complete structure of the enzyme must be determined. The first step in this process is the complete determination of its composition. X-ray crystallographic analysis at high resolution was required in addition to chemical analysis for crystalline enzyme preparation. 

In studies of catalase, much effort has been directed toward a determination of whether or not hydrogen peroxide could be dissociated from the enzyme-substrate intermediates of catalases and peroxidases. It should be pointed out that catalase, as contrasted with cytochrome oxidase, has been studied only at room temperature, and if any lesson is to be learned from the study of cytochrome oxidase 150), it is that the complexes are most likely to be identified at low temperatures, as precursors of the compounds. In this sense, they are of first importance and not to be ignored in our understanding of the mechanism of enzymic reactions. 

One of the most important findings of investigations on the reaction mechanism of cytochrome c oxidase is the discovery of its proton pumping... 

Reconstitution of membranes from a small number of molecular components provides simplified structures to study. Thus, cytochrome oxidase or photosynthetic reaction centers, both electron transfer proteins, may be extracted from their native membranes, purified, and reincorporated at relatively high concentration into a simple well defined lipid bilayer. Diffraction investigation then provides information about the distribution and structure of the protein in the membrane. Understanding the mechanism for electron transport in these proteins will require considerable additional information. One key element of structural informations is the location of the redox centres in the membrane profile. 

Fig. 3.3. Tentative mechanism of reduction of dioxygen. The scheme shows some of the more significant reaction steps at the haem iron-Cug centre of cytochrome oxidase. The reaction may be initiated by delivery of dioxygen to the reduced enzyme (in anaerobiosis top of figure). An initially formed oxy intermediate is normally extremely short-lived, but can be stabilised and identified in artificial conditions (see Refs. 92, 99,129, 134). Concerted transfer of two electrons from Fe and Cu to bound dioxygen yields a peroxy intermediate. This, or its electronic analogue, is stabilised in the absence of electron donors (ferrocytochrome a and/or reduced Cu ), and has been termed Compound C [129,130,132). It may also be observed at room temperature, and is then probably generated from the oxidised state by partial oxidation of water in the active site, in an energy-linked reversed electron transfer reaction [29] (see also Refs. 92, 99). Also the ferryl intermediate [92,99,100] has been tentatively observed in such conditions [29]. In aerobic steady states the reaction is thought to involve the cycle of intermediates in the centre of the figure (dark frames). The irreversible step is probably the conversion of g = 6 (see Refs. 98, 133) to peroxy .

Theoretical studies of the important class of enzyme reactions where an 0-0 bond is either formed or cleaved are described. Photosystem II is the only enzyme that can form 0-0 bonds from water, and suggested mechanisms for how this might occur axe discussed. In contrast, several enzymes are able to cleave 0-0 bonds. The main examples discussed here axe cytochrome oxidase and methane monooxygencise. Other examples described axe heme peroxidases, mangeinese catalase and isopenicillin N synthase. General features are discussed for these reactions, which are shown to usually involve spin-state changes. The appeairance of radicals and critical roles of protonations are emphasized. 

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