The roles of the two proton input channels in cytochrome c oxidase from Rhodobacter sphaeroides probed by the effects of site-directed mutations on time-resolved electrogenic intraprotein proton transfer

Abstract

The crystal structures of cytochrome c oxidase from both bovine and Paracoccus denitrificans reveal two putative proton input channels that connect the heme-copper center, where dioxygen is reduced, to the internal aqueous phase. In this work we have examined the role of these two channels, looking at the effects of site-directed mutations of residues observed in each of the channels of the cytochrome c oxidase from Rhodobacter sphaeroides. A photoelectric technique was used to monitor the time-resolved electrogenic proton transfer steps associated with the photo-induced reduction of the ferryl-oxo form of heme a3 (Fe4+ = O2-) to the oxidized form (Fe3+OH-). This redox step requires the delivery of a "chemical" H+ to protonate the reduced oxygen atom and is also coupled to proton pumping. It is found that mutations in the K channel (K362M and T359A) have virtually no effect on the ferryl-oxo-to-oxidized (F-to-Ox) transition, although steady-state turnover is severely limited. In contrast, electrogenic proton transfer at this step is strongly suppressed by mutations in the D channel. The results strongly suggest that the functional roles of the two channels are not the separate delivery of chemical or pumped protons, as proposed recently [Iwata, S., Ostermeier, C., Ludwig, B. & Michel, H. (1995) Nature (London) 376, 660-669]. The D channel is likely to be involved in the uptake of both "chemical" and "pumped" protons in the F-to-Ox transition, whereas the K channel is probably idle at this partial reaction and is likely to be used for loading the enzyme with protons at some earlier steps of the catalytic cycle. This conclusion agrees with different redox states of heme a3 in the K362M and E286Q mutants under aerobic steady-state turnover conditions.

Figure 1

Absolute absorption spectra of the wild-type and mutant forms of COX in the aerobic steady state with ascorbate + TMPD as electron donors. The stirred 1-cm cell contained 0.2–0.5 μM of wild-type or mutant COX in 50 mM potassium phosphate (pH 8.0) and 0.1% lauryl maltoside. The spectra shown were recorded about 3 min after the addition of 1 mM ascorbate and 100 μM TMPD. KCN (1 mM) and then solid dithionite were added subsequently to obtain full reduction of heme a. The spectra have been normalized by the amplitude of the peak of heme a at 605 nm vs. the 630-nm reference point in the dithionite-reduced form of the enzyme. Slit width = 2 nm. (A) Soret band spectra. (B) Visible spectra. Inset in A gives a difference between the normalized absolute “steady-state” spectra of the E286Q and K362M mutants.

Figure 2

Rapid kinetics of Δψ generation by the wild-type R. sphaeroides COX. The reaction mixture contains 5 mM Tris-acetate buffer (pH 8), 40 μM RuBpy as photoreductant of COX, and 10 mM aniline as sacrificial electron donor to the photooxidized RuBpy. Before the flash, the sample was incubated for several minutes with 1 mM H₂O₂ to convert heme a₃ to the ferryl-oxo state. After recording a trace (Wildtype), 1 mM potassium cyanide was added and the second trace (+KCN) was recorded. Inset shows a typical photoelectric trace as observed with bovine heart COX under the same conditions except that the H₂O₂ concentration was 4 mM.

Figure 3

Effect of mutations in the K channel and the D channel on the membrane potential generation by R. sphaeroides COX following single-electron photoreduction. Where shown on the same panel, the mutant and wild-type traces have been normalized for clarity to the amplitude of the microsecond phase. (A) E286Q. Basic conditions, same as in Fig. 2, except that 0.2 mM ferricyanide has been added in case of E286Q to keep heme a oxidized. (B) D132N. Basic conditions, as in case of E286Q. Where indicated, 1 mM KCN has been added. (C) T359A. Basic conditions, as in Fig. 2. (D) K362M. Basic conditions, as in Fig. 2. The traces (–4) have been recorded in sequence as follows. Traces: 1, no additions; 2, 5-min incubation with 0.2 mM ferricyanide; 3, addition of 1 mM H₂O₂; 4, addition of 0.5 mM KCN.

Figure 4

Possible roles of the two input proton channels in cytochrome oxidase. Two proposed states of cytochrome oxidase are illustrated, differing with respect to which of the proton-conducting channels is operational. On the left is the preloading or eu-oxidase conformation, when the K channel appears to be dominant. (Right) The D channel dominates during the second (peroxidase) portion of the catalytic cycle, during which proton pumping occurs.