Blocking the K-pathway still allows rapid one-electron reduction of the binuclear center during the anaerobic reduction of the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides

Abstract

The K-pathway is one of the two proton-input channels required for function of cytochrome c oxidase. In the Rhodobacter sphaeroides cytochrome c oxidase, the K-channel starts at Glu101 in subunit II, which is at the surface of the protein exposed to the cytoplasm, and runs to Tyr288 at the heme a3/CuB active site. Mutations of conserved, polar residues within the K-channel block or inhibit steady state oxidase activity. A large body of research has demonstrated that the K-channel is required to fully reduce the heme/Cu binuclear center, prior to the reaction with O2, presumably by providing protons to stabilize the reduced metals (ferrous heme a3 and cuprous CuB). However, there are conflicting reports which raise questions about whether blocking the K-channel blocks both electrons or only one electron from reaching the heme/Cu center. In the current work, the rate and extent of the anaerobic reduction of the heme/Cu center were monitored by optical and EPR spectroscopies, comparing the wild type and mutants that block the K-channel. The new data show that when the K-channel is blocked, one electron will still readily enter the binuclear center. The one-electron reduction of the resting oxidized ("O") heme/Cu center of the K362M mutant, results in a partially reduced binuclear center in which the electron is distributed about evenly between heme a3 and CuB in the R. sphaeroides oxidase. Complete reduction of the heme/Cu center requires the uptake of two protons which must be delivered through the K-channel.

Figure 1

The active site and residues in the proton-input pathways of the cytochrome c oxidase from R. sphaeroides. Red spheres are water molecules that are resolved in the X-ray structures.

Figure 2

Kinetics of the anaerobic reduction of the hemes in the wild type (WT) and K362M mutant oxidases. Stopped-flow measurements were recorded at 25°C using a 1 cm path length. The absorption difference [A_445nm-A_460nm] was divided by the nominal enzyme concentration to give an operational extinction coefficient, ε_445–460 (μM⁻¹cm⁻¹). The buffer used was 50 mM bis-Tris propane, pH 8, with 0.05% dodecylmaltoside. One syringe contained 4 mM dithionite, and enzyme was contained in the second syringe. (A) The kinetics of reduction of K362M was collected over two time ranges, t = 20 sec (solid) and t = 1000 sec (dotted). (B)The top panel shows the reduced spectrum of K362M recorded at t = 5 sec (solid) and t = 1000 sec (dotted). The bottom panel shows the resolved component spectra of the reduction kinetics. The largest component with Soret absorption at 425 and 444 nm corresponds to heme a [31] and has a rate of 2 s⁻¹. The second and third components, with troughs at 413 nm, correspond to heme a₃ [31] and have rates of 0.37 s⁻¹ and 0.022 s⁻¹ respectively.

Figure 3

X-band EPR spectra of (A) 90 μM oxidized K362M oxidase; (B) K362M oxidase reduced with 2mM dithionite for 5sec and then flash frozen; (C) wild type oxidase reduced with 2mM dithionite for 5sec and then flash frozen; (D) myoglobin, used as a standard to quantify the concentration of high-spin heme. Conditions: temperature, 15K; microwave power, 20mW; modulation amplitude, 10G; microwave frequency, 9.0448 GHz.

Figure 4

Kinetics of the anaerobic reduction of the heme groups in the K362M and K362T mutant oxidases. Conditions were the same as described in the legend to Figure 2, with the exception that the syringe containing the dithionite also contained 100μM hexaammine ruthenium. The reduction of heme a is virtually complete within the dead time of the instrument.