Electron bifurcation arises from emergent features of multicofactor enzymes

Quinone-based electron bifurcation (EB) catalyzed by cytochrome bc ₁ (cyt bc ₁ ) plays a critical role in maximizing efficiency of biological energy conversion. The canonical EB model (CEB), grounded in equilibrium redox potentials, dictates the order of EB steps with initial endergonic reduction of high-potential iron-sulfur cluster (2Fe2S) by quinol followed by exergonic reduction of low-potential heme b _L ( b _L ) by semiquinone (SQ). However, this concept falls short in explaining several experimental observations, including intermediate semiquinone spin-coupled to 2Fe2S (SQ-2Fe2S ^red ) and the absence of short-circuiting. Presented here DFT calculations on large cluster models of cyt bc ₁ , encompassing both 2Fe2S and b _L , identified location of donor (HOMO) and acceptor (LUMO) orbitals along with the previously not considered microstates to reveal that EB is an emergent property of an integrated system of redox cofactors where transient charge separations dynamically modulate electron affinities. In this system, electron transfer initiates preferentially toward b _L , indicating a departure from the conventional sequence proposed by CEB. Based on this finding, we introduce an EMBER (EMergent B _L -first Electron Routing) model of EB and demonstrate that its assumptions are supported by electron paramagnetic resonance spectroscopy data. Unlike CEB, EMBER proposes a relatively flat energy profile for EB that accommodates stable SQ-2Fe2S ^red and explains suppression of short-circuits without additional assumptions. It highlights the importance of state-dependent electrostatic interactions in shaping electron transfer pathways. In general, the concept of emergence inherent to EMBER offers a mechanistic framework applicable to a broad range of multi-cofactor redox enzymes beyond cyt bc ₁ .