Frank G. M. Wiertz

An oxo-ferryl tryptophan radical catalytic intermediate in cytochrome c and quinol oxidases trapped by microsecond freeze-hyperquenching (MHQ)

The pre‐steady state reaction kinetics of the reduction of molecular oxygen catalyzed by fully reduced cytochrome oxidase from Escherichia coli and Paracoccus denitrificans were studied using the newly developed microsecond freeze‐hyperquenching mixing‐and‐sampling technique. Reaction samples are prepared 60 and 200 μs after direct mixing of dioxygen with enzyme. Analysis of the reaction samples by low temperature UV–Vis spectroscopy indicates that both enzymes are trapped in the PM state. EPR spectroscopy revealed the formation of a mixture of two radicals in both enzymes. Based on its apparent g‐value and lineshape, one of these radicals is assigned to a weakly magnetically coupled oxo‐ferryl tryptophan cation radical. Implications for the catalytic mechanism of cytochrome oxidases are discussed.

Kinetic resolution of a tryptophan -radical intermediate in the reaction cycle of Paracoccus denitrificans cytochrome c oxidase

The catalytic mechanism, electron transfer coupled to proton pumping, of heme-copper oxidases is not yet fully understood. Microsecond freeze-hyperquenching single turnover experiments were carried out with fully reduced cytochrome aa3 reacting with O2 between 83 μs and 6 ms. Trapped intermediates were analyzed by low temperature UV-visible, X-band, and Q-band EPR spectroscopy, enabling determination of the oxidation-reduction kinetics of CuA, heme a, heme a3, and of a recently detected tryptophan radical (Wiertz, F. G. M., Richter, O. M. H., Cherepanov, A. V., MacMillan, F., Ludwig, B., and de Vries, S. (2004) FEBS Lett. 575, 127-130). CuB and heme a3 were EPR silent during all stages of the reaction. CuA and heme a are in electronic equilibrium acting as a redox pair. The reduction potential of CuA is 4.5 mV lower than that of heme a. Both redox groups are oxidized in two phases with apparent half-lives of 57 μs and 1.2 ms together donating a single electron to the binuclear center in each phase. The formation of the heme a3 oxoferryl species PR (maxima at 430 nm and 606 nm) was completed in ∼130 μs, similar to the first oxidation phase of CuA and heme a. The intermediate F (absorbance maximum at 571 nm) is formed from PR and decays to a hitherto undetected intermediate named FW*. FW* harbors a tryptophan radical, identified by Q-band EPR spectroscopy as the tryptophan neutral radical of the strictly conserved Trp-272 (Trp-272*). The Trp-272* populates to 4-5% due to its relatively low rate of formation (t½ = 1.2 ms) and rapid rate of breakdown (t½ = 60 μs), which represents electron transfer from CuA/heme a to Trp-272*. The formation of the Trp-272* constitutes the major rate-determining step of the catalytic cycle. Our findings show that Trp-272 is a redox-active residue and is in this respect on an equal par to the metallocenters of the cytochrome c oxidase. Trp-272 is the direct reductant either to the heme a3 oxoferryl species or to Cu 2+B. The potential role of Trp-272 in proton pumping is discussed.

Efficient electron transfer in a protein network lacking specific interactions.

In many biochemical processes, proteins need to bind partners amidst a sea of other molecules. Generally, partner selection is achieved by formation of a single-orientation complex with well-defined, short-range interactions. We describe a protein network that functions effectively in a metabolic electron transfer process but lacks such specific interactions. The soil bacterium Paracoccus denitrificans oxidizes a variety of compounds by channeling electrons into the main respiratory pathway. Upon conversion of methylamine by methylamine dehydrogenase, electrons are transported to the terminal oxidase to reduce molecular oxygen. Steady-state kinetic measurements and NMR experiments demonstrate a remarkable number of possibilities for the electron transfer, involving the cupredoxin amicyanin as well as four c-type cytochromes. The observed interactions appear to be governed exclusively by the electrostatic nature of each of the proteins. It is concluded that Paracoccus provides a pool of cytochromes for efficient electron transfer via weak, ill-defined interactions, in contrast with the view that functional biochemical interactions require well-defined molecular interactions. It is proposed that the lack of requirement for specificity in these interactions might facilitate the integration of new metabolic pathways.

Amicyanin transfers electrons from methylamine dehydrogenase to cytochrome c-551i via a ping-pong mechanism, not a ternary complex.

The first crystal structure of a ternary redox protein complex was comprised of the enzyme methylamine dehydrogenase (MADH) and two electron transfer proteins, amicyanin and cytochrome c-551i from Paracoccus denitrificans [Chen et al. Science 1994, 264, 86-90]. The arrangement of the proteins suggested possible electron transfer from the active site of MADH via the amicyanin copper ion to the cytochrome heme iron, although the distance between the metals is large. We studied the interactions between these proteins in solution. A titration followed by NMR spectroscopy shows that amicyanin binds cytochrome c-551i. The interface comprises the hydrophobic and positive patches of amicyanin, not the binding site observed in the ternary complex. NMR experiments further show that amicyanin binds tightly to MADH with an interface that matches the one observed in the crystal structure and that mostly overlaps with the binding site for cytochrome c-551i. Upon addition of cytochrome c-551i, no changes in the NMR spectrum of MADH-bound amicyanin are observed, suggesting that a possible interaction of the cytochrome with the binary complex must be very weak, with a dissociation constant higher than 2 mM. Reconstitution of the entire redox chain in vitro demonstrates that amicyanin can react rapidly with cytochrome c-551i, but that association of amicyanin with MADH inhibits this reaction. It is concluded that electron transfer from MADH to cytochrome c-551i does not involve a ternary complex but occurs via a ping-pong mechanism in which amicyanin uses the same interface for the reactions with MADH and cytochrome c-551i.

Electrical Transport Study of Phenylene‐Based π‐Conjugated Molecules in a Three‐Terminal Geometry

Abstract: We fabricated three‐terminal devices with conjugated molecules. Two different device layouts were used to measure both very short molecules (with one or two benzene rings) and relatively long ones (as long as 8 nm). To achieve an optimum gate effect, we used aluminum gates covered with a very thin native oxide layer. Molecules with thiol end groups were positioned between the source and drain electrodes by self‐assembly. The device yield was low for short molecules, most likely due to defects in the self‐assembled monolayers. Most of the devices made with short molecules did not show any gate effect at all; a small gate effect was only observed in two samples made with 1,3‐benzenedithiol. Some devices showed clear negative differential conductance peaks. In some devices made with long molecules, we observed a small change of conductance with gate voltage.

Low-temperature kinetic measurements of microsecond freeze-hyperquench (MHQ) cytochrome oxidase monitored by UV-visible spectroscopy with a newly designed cuvette.

A special cuvette was designed to measure optical changes of MHQ (microsecond freeze-hyperquench) powder samples at temperatures below approx. 250 K. Reduced cytochrome c oxidase from Paracoccus denitrificans was reacted with O(2) for 100 micros, frozen as a powder and transferred to the cuvette. Subsequently, cytochrome oxidase was allowed to react further following stepwise increments of the temperature from 100 K up to 250 K while recording spectra between 300 and 700 nm. The temperature was raised only when no further changes in the spectra could be detected. The experiment yielded spectra of the A, P(M), F and O intermediate states. This demonstrated that the catalytic cycle of cytochrome oxidase at low temperature is similar to that at room temperature and so verifies the suitability of this method for the study of enzymes with high catalytic-centre activity.