Proton-dependent electron transfer from CuA to heme a and altered EPR spectra in mutants close to heme a of cytochrome oxidase

Abstract

Eukaryotic cytochrome c oxidase (CcO) and homologous prokaryotic forms of Rhodobacter and Paraccocus differ in the EPR spectrum of heme a. It was noted that a histidine ligand of heme a (H102) is hydrogen bonded to serine in Rhodobacter (S44) and Paraccocus CcOs, in contrast to glycine in the bovine enzyme. Mutation of S44 to glycine shifts the heme a EPR signal from g(z) = 2.82 to 2.86, closer to bovine heme a at 3.03, without modifying other properties. Mutation to aspartate, however, results in an oppositely shifted and split heme a EPR signal of g(z) = 2.72/2.78, accompanied by lower activity and drastically inhibited intrinsic electron transfer from CuA to heme a. This intrinsic rate is biphasic; the proportion that is slow is pH dependent, as is the relative intensity of the two EPR signal components. At pH 8, the heme a EPR signal at 2.72 is most intense, and the electron transfer rate (CuA to heme a) is 10-130 s(-1), compared to wild-type at 90,000 s(-1). At pH 5.5, the signal at 2.78 is intensified, and a biphasic rate is observed, 50% fast (approximately wild type) and 50% slow (90 s(-1)). The data support the prediction that the hydrogen-bonding partner of the histidine ligand of heme a is one determinant of the EPR spectral difference between bovine and bacterial CcO. We further demonstrate that the heme a redox potential can be dramatically altered by a nearby carboxyl, whose protonation leads to a proton-coupled electron transfer process.

Figure 1

The structure of the two subunits of Rs CcO from 2GSM (10) is shown with subunit I (gray) containing heme a and a₃ (green sticks) and Cu_B as an orange sphere, with the metals of Ca and Mg (green spheres) close to the subunit I/II interface. Key residues are shown (sticks) on helix I (yellow ribbon): H93, H102 (a ligand of heme a), S44, and D132 at the entrance of the proton uptake path. Subunit II (red) is shown with its dinuclear Cu_A (orange spheres).

Figure 2

The ligands to heme a are shown for bovine (2DYR, oxidized structure) and Rs (2GSM, oxidized structure) CcO. The residues that are closest to the heme a ligands are shown in sticks. Bovine CcO shows a glycine (G30) with the carbonyl backbone oxygen hydrogen-bonding to His 61. Rs CcO shows a serine (S44) with its hydroxyl hydrogen-bonding to His 102.

Figure 3

The UV–vis spectra of reduced wild-type and S44D, S44G, and S44N Rs CcO. All show normal Soret (446 nm) and α (606 nm) band peaks. Buffer contained 200 mM HEPES–KOH, pH 7.4, and 0.1% dodecyl maltoside with sodium dithionite as a reductant.

Figure 4

The top two panels show the steady-state activity of S44G (△), S44D (◯), and wild-type (■) Rs CcO at different pH values over the pH range 6–9, with errors bars shown if significant. The lower two panels show the stopped-flow rates of activity, in which the cytochrome c is not saturating. The left panels show the activity (e⁻ s⁻¹ aa₃⁻¹), and the right panels show the percentage activity. The buffers MES–KOH, HEPES–KOH, and CHES–KOH were used at 50 mM with appropriate amounts of KCl to give approximate ionic strength of 45 mM with respect to potassium and with dodecyl maltoside at 0.1%.

Figure 5

Measurements of proton uptake and release by wild-type, S44G, and S44D CcO in proteoliposomes (COVs) using the externally added pH-sensitive dye, phenol red. The assays contained 0.1 μM aa₃ with pH adjusted to pH 7.4 with 50 μM HEPES–KOH on the outside and 75 mM HEPES–KOH on the inside. Increasing absorbance in the controlled state, C, indicates alkalinization on the outside of COVs as a result of proton backflow due to a high ΔΨ + ΔpH. Addition of valinomycin, V, relieves the ΔΨ, resulting in a decrease in absorbance consistent with proton pumping to the outside of the COVs. In the uncoupled state, U, addition of FCCP results in net alkalinization, as protons are consumed in the reduction of oxygen to water and equilibrate across the membrane.

Figure 6

EPR spectra of wild-type Rs CcO (black line), and mutants, S44G (red line) and S44D (blue line), with bovine g values (green) below the spectra and indicated with green arrows. The g_z, g_x, g_y values are given for the g_z, g_x, and g_y of heme a. (See also Table 2.) Samples were made in a buffer of 20 mM HEPES–KOH, pH 8.0, 14 mM KCl, and 0.1% lauryl maltoside with final concentrations of 82, 88, and 61 μM CcO, respectively. EPR experimental conditions: microwave frequency, 9.458 GHz; microwave power, 50 μW; modulation frequency, 100 kHz; modulation amplitude, 20.0 G; conversion time, 327 ms; temperature, 4.2 K.

Figure 7

(A) Overlay of the EPR spectral changes with wild-type Rs CcO (black), S44G (red), S44D (green), and S44N (blue) in 20 mM HEPES–KOH, pH 8.0, 14 mM KCl, and 0.1% lauryl maltoside. The EPR spectra were measured with a microwave frequency of 9.458 GHz, a microwave power of 50 μW, and a modulation amplitude of 20.0 G at 4.2 K. (B) Effect of pH on the amplitude of the heme a g_z EPR spectra of S44D. 50 mM MES–KOH buffer was used for pH 5.5 and 6.5 with 0.1% lauryl maltoside, whereas pH 7.4 and 8.0 buffers contained 50 mM HEPES–KOH and 0.1% lauryl maltoside. Ionic strength was controlled by adjusting the conductivity with NaCl. Colored lines are shown for pH 5.5 (black), pH 6.5 (red), pH 7.4 (green), and pH 8.0 (blue). EPR conditions are described in Methods. The changes in the split 2.72/2.78 peaks, with pH change, are depicted.

Figure 8

Ionic strength dependence of reaction of Ru-39-Cc with native CcO, S44G CcO, and S44D CcO at pH 8.0. The solutions contained 12 μM Ru-39-Cc, 15 μM CcO, 5 mM Tris-HCl, pH 8.0, 10 mM aniline, 1 mM 3CP, 0–300 mM NaCl, and 0.1% lauryl maltoside. Wild-type CcO: ○ (black), intracomplex k_a; ● (black), bimolecular k_aobs. S44G CcO: □ (blue), intracomplex k_a; ■ (blue), bimolecular k_aobs. S44D CcO: △ (red), intracomplex k_a; ▲ (red), bimolecular k_aobs; ◆ (green), k_b. Reaction of 20 μM Ru₂Z with 13 μM S44D CcO, × (magenta).

Figure 9

Photoinduced electron transfer from 12 μM Ru-39-Cc to 15 μM S44D CcO under the same conditions as in Figure 8 with 70 mM NaCl. Absorbance transients are shown at 830 and 600 nm.

Figure 10

Effect of native horse Cc on the reaction of 4.8 μM Ru-39-Cc with 5.8 μM S44D CcO under the same conditions as in Figure 8 with 70 mM NaCl. The total Cc concentration shown is the sum of the Ru-39-Cc and native Cc concentrations.

Figure 11

Ionic strength dependence of reaction of Ru-39-Cc with S44D CcO in 5 mM sodium phosphate, pH 6.5, 10 mM aniline, 1 mM 3CP, 0–300 mM NaCl, and 0.1% lauryl maltoside, following the Cc at 550 nm, Cu_A at 830 nm, and heme a at 600 nm. Key: ●, intracomplex k_a; ■, bimolecular k_aobs measured at 550 nm; ▲, fast phase of reduction of heme a measured at 600 nm; ◆, slow phase of reduction of heme a.

Figure 12

Time course of photoinduced electron transfer from Ru-39-Cc S44D CcO at pH 6.5. The conditions are the same as in Figure 11 with 70 mM NaCl over a short time scale.

Figure 13

pH dependence of reaction of Ru-39-Cc with S44D CcO in 10 mM aniline, 1 mM 3CP, 70 mM NaCl, 0.1% lauryl maltoside, and 5 mM buffer (a mixture of Tris-HCl, MES, and acetate). (●) The rate constant of the slow phase of reduction of heme a, k_b. (■) The % fast phase of reduction of heme a.

Figure 14

Time course of photoinduced electron transfer from Ru-39-Cc S44D CcO at pH 5.5, following Cu_A at 830 nm and heme a at 600 nm. The conditions are the same as in Figure 13 with 70 mM NaCl.

Scheme 1

Scheme 2