The mechanism of ubihydroquinone oxidation at the Qo-site of the cytochrome bc1 complex.

1. Recent results suggest that the major flux is carried by a monomeric function, not by an intermonomer electron flow. 2.

Role of the -PEWY-glutamate in catalysis at the Q(o)-site of the Cyt bc(1) complex.

We re-examine the pH dependence of partial processes of ubihydroquinone (QH(2)) turnover in Glu-295 mutants in Rhodobacter sphaeroides to clarify the mechanistic role. In more crippled mutants, the bell-shaped pH profile of wildtype was replaced by dependence on a single pK at ~8.5 favoring electron transfer.

Inter-monomer electron transfer is too slow to compete with monomeric turnover in bc(1) complex.

The homodimeric bc(1) complexes are membrane proteins essential in respiration and photosynthesis. The ~11Å distance between the two b(L)-hemes of the dimer opens the possibility of electron transfer between them, but contradictory reports make such inter-monomer electron transfer controversial. We have constructed in Rhodobacter sphaeroides a heterodimeric expression system similar to those used before, in which the bc(1) complex can be mutated differentially in the two copies of cyt b to test for inter-monomer electron transfer, but found that genetic recombination by cross-over then occurs to produce wild-type homodimer.

The Q-cycle reviewed: How well does a monomeric mechanism of the bc(1) complex account for the function of a dimeric complex?

Recent progress in understanding the Q-cycle mechanism of the bc(1) complex is reviewed. The data strongly support a mechanism in which the Q(o)-site operates through a reaction in which the first electron transfer from ubiquinol to the oxidized iron-sulfur protein is the rate-determining step for the overall process. The reaction involves a proton-coupled electron transfer down a hydrogen bond between the ubiquinol and a histidine ligand of the [2Fe-2S] cluster, in which the unfavorable protonic configuration contributes a substantial part of the activation barrier.

Cryoradiolytic reduction of heme proteins: Maximizing dose dependent yield.

Radiolytic reduction in frozen solutions and crystals is a useful method for generation of trapped intermediates in protein based radical reactions. In this communication we define the conditions which provide the maximum yield of one electron reduced myoglobin at 77 K using (60)Co γ-irradiation in aqueous glycerol glass. The yield reached 50% after 20 kGy, was almost complete at ∼160 kGy total dose, and does not depend on the protein concentration in the range 0.

Resonance Raman detection of the hydroperoxo intermediate in the cytochrome P450 enzymatic cycle.

[Figure: see text] The Resonance Raman spectra of the hydroperoxo complex of camphor-bound CYP101 have been obtained by cryoradiolytic reduction of the oxygenated ferrous form that had been rapidly frozen in water-glycerol frozen solution; EPR spectroscopy was employed to confirm the identity of the trapped intermediate. The ν(O-O) mode, appearing at 799cm, is observed for the first time in a peroxo-heme adduct. It is assigned unambiguously by employing isotopomeric mixtures of oxygen gas containing 50% OO, confirming the presence of an intact O-O fragment.

Characterization of humanized antibodies secreted by Aspergillus niger.

Two different humanized immunoglobulin G1(kappa) antibodies and an Fab' fragment were produced by Aspergillus niger. The antibodies were secreted into the culture supernatant. Both light and heavy chains were initially synthesized as fusion proteins with native glucoamylase.