P. Leslie Dutton

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.

Oxidation-reduction potential dependence of the interaction of cytochromes, bacteriochlorophyll and carotenoids at 77°K in chromatophores of Chromatium D and Rhodopseudomonas gelatinosa

Abstract 1. A technique for assay of light-induced reactions at 77°K as a function of oxidation-reduction potential has been developed. 2. The light-induced reactions at 77°K in Rhodopseudomonas gelatinosa and Chromatium D have been studied as a function of oxidation-reduction potential imposed on the chromatophores before freezing. 3. In Chromatium D, cytochrome C553 (Em + 10 mV) is oxidized by the bacteriochlorophyll reaction center complex (Em + 470 mV) which undergoes light-induced bleaching at 615 nm, 430 nm and 400 nm; these spectral components are probably part of the reaction center P890 (P883). The evidence suggests that either of two cytochrome C553 hemes are capable of transferring electrons to bacteriochlorophyll+ at 77°K. The primary electron acceptor associated with the reaction center bacteriochlorophyll has Em −135 mV. 4. In R. gelatinosa, cytochromes C422 (Em + 280 mV) and C419 (Em + 128 mV) are both apparently oxidized by a reaction center bacteriochlorophyll, Em + 400 mV; the molar ratio of these three components reacting at 77°K is 0.55:1:1, respectively. The primary electron acceptor of this system has Em −140 mV. However, the fact that some cytochrome oxidation was detectable at −350 mV indicates another and lower potential acceptor. 5. Evidence in the cited species indicates that a single reaction center oxidizes both the high and low potential cytochromes. 6. As the ambient oxidation-reduction potential is decreased, the control of cytochrome C422 oxidation in R. gelatinosa appears directly associated with the oxidation-reduction curve of the low potential cytochrome C419. 7. Carotenoid absorbance changes observed in R. gelatinosa at 77°K appear to respond to the light-induced oxidation-reduction reactions of reaction center bacteriochlorophyll, the primary electron acceptor, and to the cytochromes; the carotenoid changes may result from electrostatic field alterations.

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.

Cytochrome c2 and reaction center of Rhodospeudomonas spheroides Ga. membranes. Extinction coefficients, content, half-reduction potentials, kinetics and electric field alterations

The reduced minus oxidized extinction coefficients (delta epsilon-red-ox) of reaction center P605 when in the chromatophore is about 20% smaller than in the detergent-isolated state. Presumably the coupling of the reaction center protein to the antenna bacteriochlorophylls and carotenoids causes this hypochromism. The chromatophore values for P605 are 19.5 mM- minus 1 times cm- minus 1 with the spectrophotometer on single beam mode at 605 nm, and 29.8 mM- minus 1 times cm- minus 1 on dual wavelength mode set at 605--540 nm. Cytochrome c2, which is not affected by detergent, has a delta epsilon-red-ox value at 550--540 nm of 19.0 mM- minus 1 times cm- minus 1.2. The total bacteriochlorophyll to reaction center bacteriochlorophyll protein (P) ratio is about 100: 1. The cytochrome c2: reaction center protein ratio approaches 2. In current French press chromatophore preparations, about 70% of the reaction centers are each associated on a rapid kinetic basis with two cytochrome c2 molecules (intact P-c2 units). The remaining reaction center proteins are not associated with cytochrome c2 on a kinetically viable bais and may be the result of damage incurred during mechanical rupture of the cells. 3...

Energy dependent changes in the oxidation-reduction potential of cytochrome b☆

The oxidation-reduction midpoint potential measurement of mitochondrial cytochrome b indicate that there are two chemically different species of cytochrome b in the respiratory chain. The midpoint potential of one of these species is energy dependent.

Design and engineering of an O(2) transport protein.

The principles of natural protein engineering are obscured by overlapping functions and complexity accumulated through natural selection and evolution. Completely artificial proteins offer a clean slate on which to define and test these protein engineering principles, while recreating and extending natural functions. Here we introduce this method with the design of an oxygen transport protein, akin to human neuroglobin. Beginning with a simple and unnatural helix-forming sequence with just three different amino acids, we assembled a four-helix bundle, positioned histidines to bis-histidine ligate haems, and exploited helical rotation and glutamate burial on haem binding to introduce distal histidine strain and facilitate O2 binding. For stable oxygen binding without haem oxidation, water is excluded by simple packing of the protein interior and loops that reduce helical-interface mobility. O2 affinities and exchange timescales match natural globins with distal histidines, with the remarkable exception that O2 binds tighter than CO.

Reversible redox energy coupling in electron transfer chains

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 Qo site, which shows that redox equilibria are relevant on a catalytic timescale. Such rapid reversibility renders popular models based on a semiquinone in Qo 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.

Design of a heme-binding four-helix bundle

The design and characterization of two synthetic peptides that self-assemble into heme-binding proteins are described. The peptides are intended to fold into a four-helix bundle and bind a single heme parallel to the helices in the bundle core using two histidine side chains as ligands. Both proteins bind a single heme in the binding pocket. In one protein there are comparable amounts of low- and high-spin hemes, while in the other low-spin heme predominates. In both proteins, the EPR spectra of the low-spin heme indicate bis-imidazole ligation. The results illustrate that subtle differences in packing, binding pocket flexibility, and ligand orientation can significantly influence the characteristics of functionalized peptides

Primary processes in photosynthesis: Insitu ESR studies on the light induced oxidized and triplet state of reaction center bacteriochlorophyll

Summary ESR studies at liquid helium temperatures have been conducted on chromatophore and subchromatophore preparations from Chromatium D. If the primary electron acceptor of reaction center bacteriochlorophyll is chemically in a reduced state before illumination, the light activated exited state bacteriochlorophyll is prevented from undergoing oxidation. This is evidenced under these conditions by the absence of the familiar g ≅ 2 signal. Instead, a new ESR spectrum is generated in the light. This is comprised of both absorption and emission bands. The oxidation-reduction potential dependence and kinetics of the ESR changes, activated by laser pulses, suggest the signals represent bacteriochlorophyll in the triplet state. This state could be a primary intermediate in the early light activated transitions of photosynthesis.

Electrochemistry of ubiquinones: Menaquinones and plastoquinones in aprotic solvents

First and second half‐wave reduction potentials of a series of 1,4‐benzo‐ and 1,4‐naphtho‐quinones related to the naturally occurring ubiquinones, plastoquinones and menaquinones are correlated with substituent effects. Notably, E of 2,3‐dimethoxy‐1,4‐benzoquinone is positive of the values for the 2,5‐ and 2,6‐dimethoxy isomers, and of the value for methoxy‐1,4‐benzoquinone. This phenomenon is attributed to steric inhibition of resonance when two methoxy groups occupy adjacent positions, and the significance of this orientation in the ubiquinone series is highlighted.