BBA - Bioenergetics (v.1757, #4)

Protein film voltammetry, the direct electrochemistry of redox enzymes and proteins, provides precise and comprehensive information on complicated reaction mechanisms. By controlling the driving force for a reaction (using the applied potential) and monitoring the reaction in real time (using the current), it allows thermodynamic and kinetic information to be determined simultaneously. Two challenges are inherent to protein film voltammetry: (i) to adsorb the protein or enzyme in a native and active configuration on the electrode surface, and (ii) to understand and interpret voltammetric results on both a qualitative and quantitative level, allowing mechanistic models to be proposed and rigorous experiments to test these models to be devised. This review focuses on the second of these two challenges. It describes how to use protein film voltammetry to derive mechanistic and biochemically relevant information about redox proteins and enzymes, and how to evaluate and interpret voltammetric results. Selected key studies are described in detail, to illustrate their underlying principles, strategies and physical interpretations.
Keywords: Electrocatalysis; Electrochemistry; Protein film voltammetry; Coupled electron transfer; Redox protein;

Reduction of nitric oxide in bacterial nitric oxide reductase—a theoretical model study by L. Mattias Blomberg; Margareta R.A. Blomberg; Per E.M. Siegbahn (240-252).
The mechanism of the nitric oxide reduction in a bacterial nitric oxide reductase (NOR) has been investigated in two model systems of the heme-b 3-FeB active site using density functional theory (B3LYP). A model with an octahedral coordination of the non-heme FeB consisting of three histidines, one glutamate and one water molecule gave an energetically feasible reaction mechanism. A tetrahedral coordination of the non-heme iron, corresponding to the one of CuB in cytochrome oxidase, gave several very high barriers which makes this type of coordination unlikely. The first nitric oxide coordinates to heme b 3 and is partly reduced to a more nitroxyl anion character, which activates it toward an attack from the second NO. The product in this reaction step is a hyponitrite dianion coordinating in between the two irons. Cleaving an N―O bond in this intermediate forms an FeB (IV)=O and nitrous oxide, and this is the rate determining step in the reaction mechanism. In the model with an octahedral coordination of FeB the intrinsic barrier of this step is 16.3 kcal/mol, which is in good agreement with the experimental value of 15.9 kcal/mol. However, the total barrier is 21.3 kcal/mol, mainly due to the endergonic reduction of heme b 3 taken from experimental reduction potentials. After nitrous oxide has left the active site the ferrylic FeB will form a μ-oxo bridge to heme b 3 in a reaction step exergonic by 45.3 kcal/mol. The formation of a quite stable μ-oxo bridge between heme b 3 and FeB is in agreement with this intermediate being the experimentally observed resting state in oxidized NOR. The formation of a ferrylic non-heme FeB in the proposed reaction mechanism could be one reason for having an iron as the non-heme metal ion in NOR instead of a Cu as in cytochrome oxidase.
Keywords: Nitric oxide reductase; NOR; Heme-copper oxidase; Nitrous oxide; Nitric oxide; DFT; B3LYP;

Effect of bicarbonate on the water-oxidizing complex of photosystem II in the super-reduced S-states by Dmitriy N. Shevela; Andrew A. Khorobrykh; Vyacheslav V. Klimov (253-261).
It is shown that the hydrazine-induced transition of the water-oxidizing complex (WOC) to super-reduced S-states depends on the presence of bicarbonate in the medium so that after a 20 min treatment of isolated spinach thylakoids with 3 mM NH2NH2 at 20 °C in the CO2/HCO3 -depleted buffer the S-state populations are: 42% of S−3, 42% of S−2, 16% of S−1 and even formal S−4 state is reached, while in the presence of 2 mM NaHCO3, the same treatment produces 30% of S−3, 38% of S−2, and 32% of S−1 and there is no indication of the S−4 state. Bicarbonate requirement for the oxygen-evolving activity, very low in untreated thylakoids, considerably increases upon the transition of the WOC to the super-reduced S-states, and the requirement becomes low again when the WOC returns back to the normal S-states using pre-illumination. The results are discussed as a possible indication of ligation of bicarbonate to manganese ions within the WOC.
Keywords: S-state; Bicarbonate; Water-oxidizing complex; Photosystem II;

Mitochondrial dysfunction in patients with severe sepsis: An EPR interrogation of individual respiratory chain components by Dimitri A. Svistunenko; Nathan Davies; David Brealey; Mervyn Singer; Chris E. Cooper (262-272).
Electron paramagnetic resonance (EPR) spectra of complex biological systems contain information about the paramagnetic centres present. Retrieving such information is important since paramagnetic species are common intermediates of all redox reactions in both normal and abnormal metabolism. However, it is often difficult to determine the nature and content of all paramagnetic species present because the EPR signals from individual centres overlap. Here, we apply our deconvolution method based on spectra subtraction with variable coefficient to quantify individual paramagnetic components of human muscle biopsies taken from critically ill patients with severe sepsis. We use low temperature EPR spectroscopy to identify and quantify nine different paramagnetic species in the tissue. These include the majority of the mitochondrial iron–sulfur centres and the first in vivo report of a mitochondrial radical assigned to a spin-coupled pair of semiquinones (SQ·–SQ·). We have previously demonstrated in these same muscle biopsies that biochemical assays of mitochondrial dysfunction correlate with clinical outcomes (D. Brealey, M. Brand, I. Hargreaves, S. Heales, J. Land, R. Smolenski, N.A. Davies, C.E. Cooper, M. Singer, Association between mitochondrial dysfunction and severity and outcome of septic shock. Lancet 360 (2002) 219–223.). Analysis of the paramagnetic centres in the muscle confirms and extends these findings: the (SQ·–SQ·) radical species negatively correlates with the illness severity of the patient (APACHE II score) and a decreased concentration of mitochondrial Complex I iron–sulfur redox centres is linked to mortality.
Keywords: Mitochondria; EPR; Free radical; Sepsis; Iron sulfur; Complex I; Muscle;

The kinetics of the cytochrome (cyt) components of the bc 1 complex (ubiquinol: cytochrome c oxidoreductase, Complex III) are traditionally followed by using the difference of absorbance changes at two or more different wavelengths. However, this difference-wavelength (DW) approach is of limited accuracy in the separation of absorbance changes of components with overlapping spectral bands. To resolve the kinetics of individual components in Rhodobacter sphaeroides chromatophores, we have tested a simplified version of a least squares (LS) analysis, based on measurement at a minimal number of different wavelengths. The success of the simplified LS analysis depended significantly on the wavelengths used in the set. The “traditional” set of 6 wavelengths (542, 551, 561, 566, 569 and 575 nm), normally used in the DW approach to characterize kinetics of cyt c tot (cyt c 1  + cyt c 2), cyt b L, cyt b H, and P870 in chromatophores, could also be used to determine these components via the simplified LS analysis, with improved resolution of the individual components. However, this set is not sufficient when information about cyts c 1 and c 2 is needed. We identified multiple alternative sets of 5 and 6 wavelengths that could be used to determine the kinetics of all 5 components (P870 and cyts c 1, c 2, b L, and b H) simultaneously, with an accuracy comparable to that of the LS analysis based on a full set of wavelengths (1 nm intervals). We conclude that a simplified version of LS deconvolution based on a small number of carefully selected wavelengths provides a robust and significant improvement over the traditional DW approach, since it accounts for spectral interference of the different components, and uses fewer measurements when information about all five individual components is needed. Using the simplified and complete LS analyses, we measured the simultaneous kinetics of all cytochrome components of bc 1 complex in the absence and presence of specific inhibitors and found that they correspond well to those expected from the modified Q-cycle. This is the first study in which the kinetics of all cytochrome and reaction center components of the bc 1 complex functioning in situ have been measured simultaneously, with full deconvolution over an extended time range.
Keywords: bc 1 complex; Electron transfer; Spectral deconvolution; Kinetics; Least squares; Rhodobacter sphaeroides;