oxidase studied by ATR-FTIR spectroscopy
4.4.7 Possible candidates for the third component
Carboxyl group amino acid candidates that could give rise to the third component are shown in Figure 4-5, E242 and D51 are also shown. These residues were identified as being functionally important by Rich, P. R. and Maréchal, A. (2008) [206]. They include carboxylic acids (D369, D364 and E198) close to the Mg2+ site, since that is where the proton trap site is located, that receives translocated protons from the D channel via E242 (if that is operative as a translocating pathway in mitochondrial CcOs - see main introduction). These residues are also close to the BNC whose redox transitions have been assigned to give rise to this third component. D91 in subunit I is at the entrance of the D channel and in crystal structures a nearby histidine residue (H503) is shown to undergo a redox linked conformational change that is proposed to result in D91 deprotonation upon reduction [35]. Other amino acids include D442 and E40. These are proposed to be involved in binding Na+/Ca2+ cations at the interface of subunits I and II, they are also close to the top of the H channel [38,212]. Alternatively, it could be a more remote residue on another subunit, such as E90 and D246, in subunit III since these residues are located in the central region of CcO where the dielectric strength is low, and so, they are likely to be protonated [206]. They could give rise to this signal via long range structural changes. E62 in subunit II is at the entrance of the K channel. The K channel is involved in delivering 2 out of 4 substrate protons to the BNC [83,84], and/or providing a dielectric well to stabilise transient intermediates [86]. Site-directed mutagenesis of this residue in Rba. sphaeroides CcO causes loss of turnover activity and blocks the K channel, and so is therefore suggested to be the predominant entry point for protons in the K channel [213].
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Figure 4-5. Structure of bovine CcO showing possible carboxyl group candidates (red) for the third 1740 cm-1 IR component (PDB:1V54), haem propionates are also highlighted. A. An expansion of carboxyl groups in subunit I close to the metal centres and Mg site (purple sphere). B. Remote carboxyl groups in subunits I (blue), II (green) and III (yellow).
4.5 Conclusions
In conclusion, both CN and CO ligands of CcO were efficient in separating the redox transitions of the redox centres by allowing stable MV states of CcO to be generated with controlled electrochemistry. In doing so, it was possible to couple the protonated carboxyl bands in the IR difference spectra with specific redox transitions in bovine CcO. Up to three components were identified at 1749 cm-1, 1740 cm-1 and 1737 cm-1. The 1749 cm-1 and 1737 cm-1 components have been coupled to the redox transition of CuA and haem a. The 1749 cm-1 component in the unligated-FR is
proposed to shift to 1740 cm-1 when CO or CN are ligated. The third 1740 cm-1 component has a more complicated coupling mechanism, it has been linked with the redox transitions of the CuB and haem a3 in such a way that its contribution cancels out
in the FR minus O IR difference spectrum. Its assignment to a specific residue requires further investigation and several candidates are shown in Figure 4-5. A change in extinction coefficient of E242 and the protonation change in D51 have been tentatively assigned to the 1749 cm-1 and 1737 cm-1 troughs, respectively.
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4.6 Future work
Different strategies will be needed to fully understand the role and contribution of protonated carboxyl groups in CcO reaction mechanism. The model proposed is based on a number of assumptions, and extension will require repeating these series of measurements with CN and CO in D2O. H/D exchange is expected to shift and possibly
separate the IR bands, hence providing further insight into the number of contributing components. If the IR bands are sensitive to H/D exchange it will provide confirmation that they do not arise from ester bonds of lipids. Definitive assignment of the third IR component to a protonated carboxylic group will require site-directed mutagenesis.
Yeast CcO is highly homologous to bovine CcO [26,41] and can be used to produce mutants in order to help with the assignment of the signals observed [118]. To date, mutants of different candidate carboxyl groups and surrounding residues have been produced based on a homology model of yeast CcO [41]. This will allow the definitive assignment of signals observed to specific carboxyl groups within the structure of a mitochondrial form of CcO. For example, a yeast CcO mutant, E243D, has confirmed the assignment of the equivalent 1749 cm-1 band to the conserved glutamic acid (Amandine Maréchal, personal communication). Also noteworthy is that the second 1737 cm-1 component in bovine CcO is absent from yeast CcO as is the D51 residue (replaced by S52) to which it has been assigned.
The spectra reported have been recorded of electrochemically poised CcO. Therefore they do not provide kinetic information on the appearance or disappearance of IR bands. Time resolved infrared spectroscopy has recently been developed as a technique that can monitor spectral changes, at the nano to micro second timescale, with high signal to noise [204]. This technique has been used to follow the kinetics of spectral changes in the carboxyl group region during the CO photolysis of CO bound CcO, and has in fact detected four bands [204]. The use of time resolved methods will provide yet another approach to separate IR bands in the carboxyl region (as well as other parts of the spectrum), according to their kinetics. This will provide further insight into the number of contributing components in regions of the IR spectrum where bands are overlapping and the order of appearance/disappearance of IR bands.