Mitochondrial complex III (CIII₂) and complex IV (CIV), which can associate into a higher-order supercomplex (SC III₂+IV), play key roles in respiration. However, structures of these plant complexes remain unknown. We present atomic models of CIII₂, CIV, and SC III₂+IV from Vigna radiata determined by single-particle cryoEM. The structures reveal plant-specific differences in the MPP domain of CIII₂ and define the subunit composition of CIV. Conformational heterogeneity analysis of CIII₂ revealed long-range, coordinated movements across the complex, as well as the motion of CIII₂’s iron-sulfur head domain. The CIV structure suggests that, in plants, proton translocation does not occur via the H channel. The supercomplex interface differs significantly from that in yeast and bacteria in its interacting subunits, angle of approach and limited interactions in the mitochondrial matrix. These structures challenge long-standing assumptions about the plant complexes and generate new mechanistic hypotheses.

Key enzymatic processes use the nonequilibrium error correction mechanism called kinetic proofreading to enhance their specificity. The applicability of traditional proofreading schemes, however, is limited because they typically require dedicated structural features in the enzyme, such as a nucleotide hydrolysis site or multiple intermediate conformations. Here, we explore an alternative conceptual mechanism that achieves error correction by having substrate binding and subsequent product formation occur at distinct physical locations. The time taken by the enzyme–substrate complex to diffuse from one location to another is leveraged to discard wrong substrates. This mechanism does not have the typical structural requirements, making it easier to overlook in experiments. We discuss how the length scales of molecular gradients dictate proofreading performance, and quantify the limitations imposed by realistic diffusion and reaction rates. Our work broadens the applicability of kinetic proofreading and sets the stage for studying spatial gradients as a possible route to specificity.