Functions of the hydrophilic channels in protonmotive cytochrome c oxidase

Abstract

The structures and functions of hydrophilic channels in electron-transferring membrane proteins are discussed. A distinction is made between proton channels that can conduct protons and dielectric channels that are non-conducting but can dielectrically polarize in response to the introduction of charge changes in buried functional centres. Functions of the K, D and H channels found in A1-type cytochrome c oxidases are reviewed in relation to these ideas. Possible control of function by dielectric channels and their evolutionary relation to proton channels is explored.

Keywords: charge transfer; dielectric properties; electrostatics; hydrophilic channels; proton transfer; redox proteins.

Figure 1.

Proton channels, proton wells and dielectric channels. (a) A proton channel that can conduct a current of protons from a source to a sink on opposite sides of a membrane. This passively conducts protons down an electrochemical proton gradient and so acts as an uncoupler. (b) Two incomplete channels for protons, or proton wells. Both are able to conduct protons to/from an exergonic reaction site. A protonmotive system is formed if the exergonic reaction is able to move protons actively from input well to output well against the electrochemical proton gradient and, in addition, forms a gate to prevent passive backflow of protons. (c) Two hypothetical incomplete dielectric channels, or dielectric wells that connect a buried site to aqueous surfaces. These do not act as uncouplers or generate a protonmotive force as they do not provide a link for sustained proton transfer between a source and sink. Instead, they provide a means to minimize transient local charge changes of buried reaction sites. They do not have to be able to conduct protons, though a proton well that can transfer a single proton to a ‘dead-end’ charge-linked Bohr group can provide the same charge-reducing dielectric function. For all dielectric channels/wells, any charge-induced change reverses when the site reverts to its ground state charge. Hence, they do not function directly in net PMF generation.

Figure 2.

Nomenclature used for the catalytic intermediates of CcOs. The catalytic intermediates discussed in this text refer only to different states of the oxygen-reducing BNC of CcO and do not take account of the redox states of Cu_A or haem a. Electrons are donated into the BNC from haem a. Starting with the catalytically active oxidized O state (ferric haem a₃, cupric Cu_B), the first and second electron transfers are charge-compensated by substrate proton uptake and produce the one-electron-reduced E state and the two-electron-reduced R state (ferrous haem a₃, cuprous Cu_B). These O → E → R steps are often referred to as the ‘reductive’ steps of the cycle. In the R state, ferrous haem a₃ binds O₂ to form the unstable oxyferrous A state. If further electrons are not immediately available from haem a, A rapidly converts to the relatively stable P_M state (ferryl haem a₃ (Fe⁴⁺ = O²⁻), and a neutral tyrosine radical). In the natural cycle, P_M is reduced when a further electron is available from haem a, together with a charge-compensating substrate proton, to form F (ferryl haem a₃, , tyrosine ground state). If an electron is already available haem a when A is formed, A is instead transformed into the P_R intermediate (ferryl haem a₃, , tyrosinate)—this is the case in flow-flash experiments described in the text in which fully reduced CcO (the FR state, with all four redox centres reduced) reacts with O₂. P_R is subsequently converted to F by uptake of a substrate proton. F is converted back to into the O state with a further electron from haem a together with a charge-compensating substrate proton. The P → F → O steps are often referred to as the ‘oxidative’ part of the reaction cycle. Each electron transfer into the BNC from haem a is generally (but see text) thought to be coupled to translocation of a proton across the membrane (not shown). See text for further details. Several quite different nomenclatures are now used in the primary CcO literature for these and additional intermediates; those above are most commonly used. However, these oxygen intermediates can be equated with those classically discussed in globins, P450s and peroxidases [48] as follows: A is equivalent to the oxyferrous state of globins and the equivalent ferric-superoxo ‘compound III’ of P450s and peroxidases; P_M is equivalent to the ferryl-radical ‘compound I’; F and P_R are equivalent to the ferryl ‘compound II’ with or without a charge-compensating proton, respectively.

Figure 3.

The hydrophilic channels in subunit I of A1-type CcOs. The crystal structures of subunit I of bovine (PDB code 1V54), Paracoccus denitrificans (PDB code 3HB3) and Rhodobacter sphaeroides (PDB code 1M56) CcOs were superimposed by alignment of their haem a macrocycles. The hydrophilic residues and resolved water molecules of their K, D and H channels are displayed in grey, blue and pink, respectively.

Figure 4.

The K channel in BtCcO. Component amino acids and associated water molecules (in red) are shown in relation to subunit I structure (grey; PDB code 1V54). The entry point of the K channel has been proposed to be either in the vicinity of E65 of subunit II (green; see text) or of H256 [38].

Figure 5.

The D channel in BtCcO. Component amino acids and nine resolved water molecules (in red) are shown in relation to subunit I structure (grey; PDB code 1V54).

Figure 6.

The H channel in BtCcO. Component amino acids and associated water molecules (in red) are shown in relation to subunit I structure (grey; PDB code 1V54).

Figure 7.

Interaction of core subunit I and supernumerary subunit IV in BtCcO. Helices XI (orange) and XII (blue) of subunit I (grey) of bovine CcO (PDB code: 1V54) have close contacts with the transmembrane helix of subunit IV (figure shows isoform 1, green). Haem a is located in one of the three four-helical ‘pores’ of subunit I, to which helices XI and XII contribute.