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. The reaction is endergonic, and the products are an unstable ubisemiquinone at the Q(o)-site, and the reduced iron-sulfur protein, the extrinsic mobile domain of which is now free to dissociate and move away from the site to deliver an electron to cyt c(1) and liberate the H(+). When oxidation of the semiquinone is prevented, it participates in bypass reactions, including superoxide generation if O(2) is available. When the b-heme chain is available as an acceptor, the semiquinone is oxidized in a process in which the proton is passed to the glutamate of the conserved -PEWY- sequence, and the semiquinone anion passes its electron to heme b(L) to form the product ubiquinone. The rate is rapid compared to the limiting reaction, and would require movement of the semiquinone closer to heme b(L) to enhance the rate constant. The acceptor reactions at the Q(i)-site are still controversial, but likely involve a "two-electron gate" in which a stable semiquinone stores an electron. Possible mechanisms to explain the cyt b(150) phenomenon are discussed, and the information from pulsed-EPR studies about the structure of the intermediate state is reviewed. The mechanism discussed is applicable to a monomeric bc(1) complex. We discuss evidence in the literature that has been interpreted as shown that the dimeric structure participates in a more complicated mechanism involving electron transfer across the dimer interface. We show from myxothiazol titrations and mutational analysis of Tyr-199, which is at the interface between monomers, that no such inter-monomer electron transfer is detected at the level of the b(L) hemes. We show from analysis of strains with mutations at Asn-221 that there are coulombic interactions between the b-hemes in a monomer. The data can also be interpreted as showing similar coulombic interaction across the dimer interface, and we discuss mechanistic implications.

1. Recent results suggest that the major flux is carried by a monomeric function, not by intermonomer electron flow. 2. The bifurcated reaction at the Qo-site involves sequential partial processes, - a rate limiting first electron transfer generating a semiquinone (SQ) intermediate, and a rapid second electron transfer in which the SQ is oxidized by the low potential chain. 3. The rate constant for the first step in a strongly endergonic, proton-first-then-electron mechanism, is given by a Marcus-Brønsted treatment in which a rapid electron transfer is convoluted with a weak occupancy of the proton configuration needed for electron transfer. 4. A rapid second electron transfer pulls the overall reaction over. Mutation of Glu-295 of cyt b shows it to be a key player. 5. In more crippled mutants, electron transfer is severely inhibited and the bell-shaped pH dependence of wildtype is replaced by a dependence on a single pK at ~8.5 favoring electron transfer. Loss of a pK ~6.5 is explained by a change in the rate limiting step from the first to the second electron transfer; the pK ~8.5 may reflect dissociation of QH·. 6. A rate constant (<103 s−1) for oxidation of SQ in the distal domain by heme bL has been determined, which precludes mechanisms for normal flux in which SQ is constrained there. 7. Glu-295 catalyzes proton exit through H+ transfer from QH·, and rotational displacement to delivers the H+ to exit channel(s). This opens a volume into which Q·− can move closer to the heme to speed electron transfer. 8. A kinetic model accounts well for the observations, but leaves open the question of gating mechanisms. For the first step we suggest a molecular “escapement”; for the second a molecular ballet choreographed through coulombic interactions.

Two different humanized immunoglobulin G1( ) 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. After antibody assembly, cleavage by A. niger KexB protease allowed the release of free antibody. Purification by hydrophobic charge induction chromatography proved effective at removing any antibody to which glucoamylase remained attached. Glycosylation at N297 in the Fc region of the heavy chain was observed, but this site was unoccupied on approximately 50% of the heavy chains. The glycan was of the high-mannose type, with some galactose present, and the size ranged from Hex 6 GlcNAc 2 to Hex 15 GlcNAc 2 . An aglycosyl mutant form of antibody was also produced. No significant difference between the glycosylated antibody produced by Aspergillus and that produced by mammalian cell cultures was observed in tests for affinity, avidity, pharmacokinetics, or antibody-dependent cellular cytotoxicity function.

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 799 cm -1 , is observed for the first time in a peroxo-heme adduct. It is assigned unambiguously by employing isotopomeric mixtures of oxygen gas containing 50% 16 O 18 O, confirming the presence of an intact O−O fragment. The ν(Fe−O) mode is observed at 559 cm -1 (H2O). Furthermore, both modes shift down by 3 cm -1 , documenting the formulation as a hydroperoxo complex, in agreement with EPR data.NOT THE PUBLISHED VERSION; this is the author's final, peer-reviewed manuscript. The published version may be accessed by following the link in the citation at the bottom of the page. Society, Vol 129, No. 20 (2007): pg. 6382-6383. DOI. This article is © American Chemical Society and permission has been granted for this version to appear in e-Publications@Marquette. American Chemical Society does not grant permission for this article to be further copied/distributed or hosted elsewhere without the express permission from American Chemical Society. Journal of the American Chemical 3The cytochromes P450 heme-thiolate enzymes facilitate quite difficult chemical transformations through a multistep reaction cycle that culminates in the generation of a remarkably potent oxidizing species capable of hydroxylating even inert substrates. [1][2][3] Following substrate binding to the resting state ferric enzyme, two sequential one-electron reductions bracketing the binding of molecular oxygen and a subsequent proton delivery step lead to heterolytic O−O bond cleavage and formation of a highly reactive ferryl heme species comparable to the so-called Compound I intermediate of peroxidases. [4][5][6] Thus, key precursors to this critical cleavage reaction are activated heme-bound peroxo and hydroperoxo fragments; that is, (protoporphyrin)Fe(III)(O2 2-) or Fe(III)(O−OH -). A useful approach to access and study these species is to generate and trap the relatively stable oxy-ferrous P450 complex and then to subject the cryotrapped (77 K) sample to radiolytic reduction using radiation from synchrotron, 60 Co γ-ray, or 32 P sources. 7,8 Enzymatic intermediates produced by cryoradiolytic reduction of the oxygenated complex of cytochrome P450cam (CYP101) have been detected by both electronic absorption and EPR spectroscopic methods. 7,9,10 However, critical mechanistic information has been missing, and it is now important to attempt to provide more detailed structural characterization of the active sites of these species.Resonance Raman (RR) spectroscopy is an especially attractive probe of such species, effectively interrogating both the heme macrocycle structure and various iron−ligand fragments. [11][12][13][14][15] In fact, the feasibility of coupling this powerful spectroscopic probe with cryogenic ...