Christopher C. Page

Natural engineering principles of electron tunnelling in biological oxidation–reduction

We have surveyed proteins with known atomic structure whose function involves electron transfer; in these, electrons can travel up to 14 Å between redox centres through the protein medium. Transfer over longer distances always involves a chain of cofactors. This redox centre proximity alone is sufficient to allow tunnelling of electrons at rates far faster than the substrate redox reactions it supports. Consequently, there has been no necessity for proteins to evolve optimized routes between redox centres. Instead, simple geometry enables rapid tunnelling to high-energy intermediate states. This greatly simplifies any analysis of redox protein mechanisms and challenges the need to postulate mechanisms of superexchange through redox centres or the maintenance of charge neutrality when investigating electron-transfer reactions. Such tunnelling also allows sequential electron transfer in catalytic sites to surmount radical transition states without involving the movement of hydride ions, as is generally assumed. The 14 Å or less spacing of redox centres provides highly robust engineering for electron transfer, and may reflect selection against designs that have proved more vulnerable to mutations during the course of evolution.

P450 BM3: the very model of a modern flavocytochrome

Flavocytochrome P450 BM3 is a bacterial P450 system in which a fatty acid hydroxylase P450 is fused to a mammalian-like diflavin NADPH-P450 reductase in a single polypeptide. The enzyme is soluble (unlike mammalian P450 redox systems) and its fusion arrangement affords it the highest catalytic activity of any P450 mono-oxygenase. This article discusses the fundamental properties of P450 BM3 and how progress with this model P450 has affected our comprehension of P450 systems in general.

Biological electron transfer

Many oxidoreductases are constructed from (a) local sites of strongly coupled substrate-redox cofactor partners participating in exchange of electron pairs, (b) electron pair/single electron transducing redox centers, and (c) nonadiabatic, long-distance, single-electron tunneling between weakly coupled redox centers. The latter is the subject of an expanding experimental program that seeks to manipulate, test, and apply the parameters of theory. New results from the photosynthetic reaction center protein confirm that the electronic-tunneling medium appears relatively homogeneous, with any variances evident having no impact on function, and that control of intraprotein rates and directional specificity rests on a combination of distance, free energy, and reorganization energy. Interprotein electron transfer between cytochromec and the reaction center and in lactate dehydrogenase, a typical oxidoreductase from yeast, are examined. Rates of interprotein electron transfer appear to follow intraprotein guidelines with the added essential provision of binding forces to bring the cofactors of the reacting proteins into proximity.

Darwin at the molecular scale: selection and variance in electron tunnelling proteins including cytochrome c oxidase.

Biological electron transfer is designed to connect catalytic clusters by chains of redox cofactors. A review of the characterized natural redox proteins with a critical eye for molecular scale measurement of variation and selection related to physiological function shows no statistically significant differences in the protein medium lying between cofactors engaged in physiologically beneficial or detrimental electron transfer. Instead, control of electron tunnelling over long distances relies overwhelmingly on less than 14 Å spacing between the cofactors in a chain. Near catalytic clusters, shorter distances (commonly less than 7 Å) appear to be selected to generate tunnelling frequencies sufficiently high to scale the barriers of multi-electron, bond-forming/-breaking catalysis at physiological rates. We illustrate this behaviour in a tunnelling network analysis of cytochrome c oxidase. In order to surmount the large, thermally activated, adiabatic barriers in the 5–10 kcal mol−1 range expected for H+ motion and O2 reduction at the binuclear centre of oxidase on the 103–105 s−1 time-scale of respiration, electron access with a tunnelling frequency of 109 or 1010 s−1 is required. This is provided by selecting closely placed redox centres, such as haem a (6.9 Å) or tyrosine (4.9 Å). A corollary is that more distantly placed redox centres, such as CuA, cannot rapidly scale the catalytic site barrier, but must send their electrons through more closely placed centres, avoiding direct short circuits that might circumvent proton pumping coupled to haems a to a3 electron transfer. The selection of distances and energetic barriers directs electron transfer from CuA to haem a rather than a3, without any need for delicate engineering of the protein medium to ‘hard wire’ electron transfer. Indeed, an examination of a large number of oxidoreductases provides no evidence of such naturally selected wiring of electron tunnelling pathways.

Simple redox-linked proton-transfer design: new insights from structures of quinol-fumarate reductase

The mitochondrial bioenergetics field has experienced an exciting breakthrough with the recent structure determination of several key membrane complexes. The latest addition to this line of structures, that of quinol-fumarate reductase, provides new insights into the mechanism of energy transduction.

Distance metrics for heme protein electron tunneling.

There is no doubt that distance is the principal parameter that sets the order of magnitude for electron-tunneling rates in proteins. However, there continue to be varying ways to measure electron-tunneling distances in proteins. This distance uncertainty blurs the issue of whether the intervening protein medium has been naturally selected to speed or slow any particular electron-tunneling reaction. For redox cofactors lacking metals, an edge of the cofactor can be defined that approximates the extent in space that includes most of the wavefunction associated with its tunneling electron. Beyond this edge, the wavefunction tails off much more dramatically in space. The conjugated porphyrin ring seems a reasonable edge for the metal-free pheophytins and bacteriopheophytins of photosynthesis. For a metal containing redox cofactor such as heme, an appropriate cofactor edge is more ambiguous. Electron-tunneling distance may be measured from the conjugated heme macrocycle edge or from the metal, which can be up to 4.8 A longer. In a typical protein medium, such a distance difference normally corresponds to a approximately 1000 fold decrease in tunneling rate. To address this ambiguity, we consider both natural heme protein electron transfer and light-activated electron transfer in ruthenated heme proteins. We find that the edge of the conjugated heme macrocycle provides a reliable and useful tunneling distance definition consistent with other biological electron-tunneling reactions. Furthermore, with this distance metric, heme axially- and edge-oriented electron transfers appear similar and equally well described by a simple square barrier tunneling model. This is in contrast to recent reports for metal-to-metal metrics that require exceptionally poor donor/acceptor couplings to explain heme axially-oriented electron transfers.

Respiratory Electron Transfer Chains

Here we examine the classical electron transfer chains of the mitochondrial respiratory oxidoreductase complexes. These chains guide electrons between substrates and energy coupling sites and between the coupling sites themselves. Structural studies on oxidoreductases are showing that several redox cofactors separated in protein by 4–10 A are often in the form of a chain Long chains are evident in respiratory Complex I, II and in between Complexes III and IV. Calculations show that these chains are able to transfer electrons over very large distances with high directional specificity. Driven by small overall free energies, they operate in the microsecond time scale. Close proximity of the cofactors maintains physiologically productive rates even when substantial thermodynamically unfavorable steps are encountered in the chain. These steps offer potential points of regulation.

Intraprotein Electron Transfer

Photosynthetic reaction centers have provided the best system in which to study natural intraprotein electron transfer. With an array of colorful redox centers conveniently activated by light, the reaction center provides access to a network of reactions over the variety of distances and free energies that has made it possible to understand the basics of design of natural electron transfer systems [1-3]. Indeed, it first became clear that tunneling is central to biological electron transfer after the observation that the photochemistry of the reaction center operates even at liquid helium temperatures [4].