Direct localization of the 51 and 24 kDa subunits of mitochondrial complex I by three-dimensional difference imaging

Abstract

Complex I is the largest complex in the respiratory chain, and the least understood. We have determined the 3D structure of complex I from Yarrowia lipolytica lacking the flavoprotein part of the N-module, which consists of the 51 kDa (NUBM) and the 24 kDa (NUHM) subunits. The reconstruction was determined by 3D electron microscopy of single particles. A comparison to our earlier reconstruction of the complete Y. lipolytica complex I clearly assigns the two flavoprotein subunits to an outer lobe of the peripheral arm of complex I. Localizing the two subunits allowed us to fit the X-ray structure of the hydrophilic fragment of complex I from Thermus thermophilus. The fit that is most consistent with previous immuno-electron microscopic data predicts that the ubiquinone reducing catalytic center resides in the second peripheral lobe, while the 75 kDa subunit is placed near the previously seen connection between the peripheral arm and the membrane arm protrusions.

Figure 1

The structure of the subcomplex was calculated from a random conical data set. Shown is a tilt pair. left: 0° image, right: tilt image, angle 55°. Tilt axis vertical. Scale bar 100 nm.

Figure 2

Surface representation of the three-dimensional reconstructions of the five classes of the subcomplex data. Class 1 and 2 show the volumes with the least differences to the complete enzyme, calculated from a total of 45% of the data. Classes 3–5 show additional conformation changes. Scale bar 10 nm.

Figure 3

a) Surface representation of the three-dimensional structure of the subcomplex, b) surface representation of the holoenzyme. The numbering of the domains follows the conventions used in the original publication of the holoenzyme structure (Radermacher et al. 2006). The major domain missing in the subcomplex is domain 1. c) added to indicate the domains not visible in a and b. Scale bar 10 nm.

Figure 4

a) overlay of surface of the holoenzyme (magenta), the subcomplex (grey) and the ribbon presentation of the X-ray structure from (Sazanov and Hinchliffe 2006), rear view. b) Surface of the subcomplex and surface of the bacterial Nqo1 (51kDa) and Nqo2 (24 kDa) subunits from the X-ray structure, represented as surface, front view. Scale bars 10 nm.

Figure 5

The five possible fits of the X-ray model into the EM structure. For better visibility the ribbon representation of the X-ray model is shown in panel 0) Indicated are the different subunits (color coded), the location of iron-sulfur cluster N2 and, marked by “*”, the loop that contains the binding epitope for antibody 49.2 in (Zickermann et al. 2003). The numbering of the panels follows the numbering of the fits as described in the text and in table 1. Scale bar 10 nm (applicable to panels 1–5)

Figure 6

Views of fit1 (left column) and fit 2 (right column). The fits are shown with the mesh data of the EM structure that fit best: the 17 Å structure in fit 1 and the 24 Å structure for fit 2. a) rear view, b) front view, c) different views for each of the two fits, for better visibility. H1 amphiphatic helix in PSST. Scale bar 100 Å, applies to panels 1–5.

Figure 7

Location of the iron sulfur clusters within the holoenzyme. a) positions for fit 1, b) positions for fit 2. In fit 1 (a) the distance between iron-sulfur cluster N2 and the upper border of the membrane arm is about 60 Å. In fit 2 (b) this distance is about 35 Å. Scale bar 10 nm.