Reversibility is a common theme in respiratory and photosynthetic systems that couple electron transfer with a transmembrane proton gradient driving ATP production. This includes the intensely studied cytochrome bc1, which catalyses electron transfer between quinone and cytochrome c. To understand how efficient reversible energy coupling works, here we have progressively inactivated individual cofactors comprising cytochrome bc1. We have resolved millisecond reversibility in all electron-tunnelling steps and coupled proton exchanges, including charge-separating hydroquinone-quinone catalysis at the Q(o) site, which shows that redox equilibria are relevant on a catalytic timescale. Such rapid reversibility renders popular models based on a semiquinone in Q(o) site catalysis prone to short-circuit failure. Two mechanisms allow reversible function and safely relegate short-circuits to long-distance electron tunnelling on a timescale of seconds: conformational gating of semiquinone for both forward and reverse electron transfer, or concerted two-electron quinone redox chemistry that avoids the semiquinone intermediate altogether.

Edited by Miguel Teixeira and Ricardo LouroKeywords: Electron transfer Photosystem Evolution Cytochrome b c1 and b 6 f Bioenergetics Reactive oxygen species (ROS) Oxidative stress Protective reactions a b s t r a c tThe energy-converting redox enzymes perform productive reactions efficiently despite the involvement of high energy intermediates in their catalytic cycles. This is achieved by kinetic control: with forward reactions being faster than competing, energy-wasteful reactions. This requires appropriate cofactor spacing, driving forces and reorganizational energies. These features evolved in ancestral enzymes in a low O 2 environment. When O 2 appeared, energy-converting enzymes had to deal with its troublesome chemistry. Various protective mechanisms duly evolved that are not directly related to the enzymes' principal redox roles. These protective mechanisms involve fine-tuning of reduction potentials, switching of pathways and the use of short circuits, back-reactions and side-paths, all of which compromise efficiency. This energetic loss is worth it since it minimises damage from reactive derivatives of O 2 and thus gives the organism a better chance of survival. We examine photosynthetic reaction centres, bc 1 and b 6 f complexes from this view point. In particular, the evolution of the heterodimeric PSI from its homodimeric ancestors is explained as providing a protective back-reaction pathway. This ''sacrifice-of-efficiency-for-protection'' concept should be generally applicable to bioenergetic enzymes in aerobic environments.

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