Direct assignment of EPR spectra to structurally defined iron-sulfur clusters in complex I by double electron-electron resonance

Abstract

In oxidative phosphorylation, complex I (NADH:quinone oxidoreductase) couples electron transfer to proton translocation across an energy-transducing membrane. Complex I contains a flavin mononucleotide to oxidize NADH, and an unusually long series of iron-sulfur (FeS) clusters, in several subunits, to transfer the electrons to quinone. Understanding coupled electron transfer in complex I requires a detailed knowledge of the properties of individual clusters and of the cluster ensemble, and so it requires the correlation of spectroscopic and structural data: This has proved a challenging task. EPR studies on complex I from Bos taurus have established that EPR signals N1b, N2 and N3 arise, respectively, from the 2Fe cluster in the 75 kDa subunit, and from 4Fe clusters in the PSST and 51 kDa subunits (positions 2, 7, and 1 along the seven-cluster chain extending from the flavin). The other clusters have either evaded detection or definitive signal assignments have not been established. Here, we combine double electron-electron resonance (DEER) spectroscopy on B. taurus complex I with the structure of the hydrophilic domain of Thermus thermophilus complex I. By considering the magnetic moments of the clusters and the orientation selectivity of the DEER experiment explicitly, signal N4 is assigned to the first 4Fe cluster in the TYKY subunit (position 5), and N5 to the all-cysteine ligated 4Fe cluster in the 75 kDa subunit (position 3). The implications of our assignment for the mechanisms of electron transfer and energy transduction by complex I are discussed.

Fig. 1.

Arrangement of the redox cofactors in B. taurus complex I, based on the structure from T. thermophilus, and possible assignments of EPR signals to clusters. Left: FeS clusters in the structure of the hydrophilic arm of T. thermophilus complex I [2FUG.PDB (2)] conserved in B. taurus, named according to their type (2Fe or 4Fe), subunit [according to the standard nomenclature for B. taurus, 75 = 75 kDa subunit, 51 = 51 kDa subunit, 24 = 24 kDa subunit, PS = PSST subunit, TY = TYKY subunit (7)], and differentiated when necessary by C (all cys ligated) or H (one his ligand), or as cluster 1 or 2. Distances are center-to-center for the FeS clusters or the central ring of the isoalloxazine system. (Right): Extant assignments of the five EPR signals observed in B. taurus complex I reduced by NADH (–6). N5 is not addressed here.

Fig. 2.

EPR spectra of complex I from B. taurus, and simulations for N1b, N2, N3, and N4. X-band CW EPR spectra of complex I reduced to -0.4 V and -0.3 V were recorded at 12 K, at 9.408 and 9.393 GHz, respectively. The W-band (93.875 GHz) echo-detected EPR spectrum (numerical 1st derivative representation) of complex I reduced to -0.4 V was recorded at 8.5 K. The three spectra (Black) are compared to one another and to their simulated spectra (18) (Red, comprising N1b, N2, N3, and N4, see Table 1), by using the g-value scale. N1b, N2, N3, and N4 are shown for the -0.4 V X-band spectrum. At -0.3 V, N1b is essentially absent. See Experimental Methods section for the measurement parameters.

Fig. 3.

The four-pulse DEER sequence. The mw detection subsequence at ω_A is applied to the detection (A) spins, and the pump pulse at frequency ω_B is applied to the pump (B) spins. The refocused echo from the detection spins at time 2(τ₁ + τ₂) is observed as a function of time (t), where t₀ = 2τ₁ is outside the spectrometer deadtime of ≈100 ns.

Fig. 4.

X-band echo-detected EPR spectrum of complex I reduced to -0.4 V recorded at 10 K and simulated with N1b, N2, N3, and N4. Data in black, simulation (see Table 1) in red. The markers correspond to the pump (Red) and detection (Black) pulse positions for the DEER traces in Fig. 5. Positions 3* and 4* refer to the -0.3 V sample; all others refer to the -0.4 V sample. See Experimental Methods section for the measurement parameters.

Fig. 5.

DEER spectra from B. taurus complex I reduced to -0.4 V and -0.3 V measured at 10 K. DEER traces (Black) and best-fit simulations (Red) for models A, B, and C. Traces 3* and 4* refer to the -0.3 V sample, all others are from the -0.4 V sample, using the following field (mT), detection (ω_A, GHz), and pump (ω_B, GHz) pulse positions (see also Fig. 4): (1) B₀ = 337.7, ω_A = 9.1104, ω_B = 9.7269; (2) B₀ = 340.5, ω_A = 9.1888, ω_B = 9.7267; (3) B₀ = 340.4, ω_A = 9.1810, ω_B = 9.4500; (3*) B₀ = 345.9, ω_A = 9.3382, ω_B = 9.5998; (4*) B₀ = 345.9, ω_A = 9.3382, ω_B = 9.5998; (5) B₀ = 347.3, ω_A = 9.3682, ω_B = 9.1888. All traces are normalized to the intensity at t = 0, and intensity changes are denoted by the scale bar. The fits shown are for k_1,2 = +1.17 and k_3,4 = -0.67; the detection-pump spin pairs used in each model are given below the figures, along with the mean intercluster distances.

Fig. 6.

Orientation selective DEER simulation parameters for a [2Fe-2S]-[4Fe-4S] pair. The unit magnetic field vector, , is defined by the angles θ and ϕ. n_ij is the unit vector between Fe ions i and j of the two clusters, r_ij is the distance between them, and is the angle between and n_ij. The g₃ axis from the [2Fe-2S] spin is along the Fe-Fe vector. The g₃ axis from the [4Fe-4S] spin is normal to the faces assigned to the Fe²⁺ and the Fe^2.5+ pairs, and, in this example, the g₁ and g₂ axes are normal to the Fe²⁺-Fe^2.5+ containing faces.