Reaction pathways in the enzymatic formation and cleavage of the N−N and N−O bonds, respectively, are difficult to verify without the structure of the intermediates, but we now have such information ...on the heme a 3 2+-NO species formed in the reaction of ba 3-oxidase with NO from resonance Raman spectroscopy. We have identified the His-heme a 3 2+-NO/CuB 1+ species by its characteristic Fe−NO and N−O stretching frequencies at 539 and 1620 cm-1, respectively. The Fe−NO and N−O frequencies in ba 3-oxidase are 21 and 7 cm-1 lower and higher, respectively, than those observed in Mb−NO. From these results and earlier Raman and FTIR measurements, we demonstrate that the protein environment of the proximal His384 that is part of the Q-proton pathway controls the strength of the Fe−His384 bond upon ligand (CO vs NO) binding. We also show by time-resolved FTIR spectroscopy that CuB 1+ has a much lower affinity for NO than for CO. We suggest that the reduction of NO to N2O by ba 3-oxidase proceeds by the fast binding of the first NO molecule to heme a 3 with high-affinity, and the second NO molecule binds to CuB with low-affinity, producing the temporal co-presence of two NO molecules in the heme-copper center. The low-affinity of CuB for NO binding also explains the NO reductase activity of the ba 3-oxidase as opposed to other heme-copper oxidases. With the identification of the His-heme a 3 2+-NO/CuB 1+ species, the structure of the binuclear heme a 3-CuB 1+ center in the initial step of the NO reduction mechanism is known.
Well-resolved, Soret band excited resonance Raman spectra were measured from the fully oxidized and fully reduced cytochrome c oxidase from beef heart and Paracoccus denitrificans. The vibrational ...patterns in the marker band region (1450-1700 cm-1) were analyzed, and a complete assignment of heme a and heme a3 vibrational modes is presented, permitting a detailed structural comparison of the mammalian and bacterial enzymes. Similar frequencies of the porphyrin modes for the reduced heme a and the reduced and oxidized heme a3 are found, indicating a close relationship of the ground-state conformations in all oxidase species studied. In oxidized heme a, however, significant frequency differences are observed and interpreted in terms of a ruffled porphyrin structure in the three- and two-subunit forms of the Paracoccus enzyme compared to the planar heme a of beef heart oxidase. The structural distortions, which also perturb the conformation of the formyl substituent and its electronic coupling with the porphyrin, reflect the specific heme-protein interactions at heme a. Since in the fully reduced state heme a appears to be largely planar in all oxidase species, the redox-linked conformational transition requires a more drastic rearrangement of the heme a-protein interactions in the bacterial than in the mammalian oxidase. For both heme a and heme a3 in the reduced state and for heme a3 in the oxidize state, frequency, intensity, and bandwidth differences of the formyl stretching vibration and intensity differences of some porphyrin modes are noted between the three oxidase forms. The same modes are also affected by quaternary structure changes in the bovine oxidase caused by different detergents and isolation procedures. These effects are attributed to differences of the dielectric properties of the heme environment, due to subtle structural changes in the heme pockets, induced by protein-protein interactions of subunit III with subunits I and/or II.
To probe the stability of the seven‐iron ferredoxin from Thermus thermophilus (FdTt), we investigated its chemical and thermal denaturation processes in solution. As predicted from the crystal ...structure, FdTt is extremely resistant to perturbation. The guanidine hydrochloride‐induced unfolding transition shows a midpoint at 6.5 m (pH 7, 20 °C), and the thermal midpoint is above boiling, at 114 °C. The stability of FdTt is much lower at acidic pH, suggesting that electrostatic interactions are important for the high stability at higher pH. On FdTt unfolding at alkaline pH, new absorption bands at 520 nm and 610 nm appear transiently, resulting from rearrangement of the cubic clusters into linear three‐iron species. A range of iron–sulfur proteins has been found to accommodate these novel clusters in vitro, although no biological function has yet been assigned.
The early observation of extra copper in preparations of cytochrome‐c oxidase has recently lead to a renewed interest in its stoichiometry and possible redox function. In various, pure preparations ...(heme A contents close to the theoretical value of 9.79 nmol/mg protein for the 13‐subunit bovine enzyme) protein‐related metal stoichiometries of 3 Cu, 2 Fe, 1 Zn, 1 Mg/monomer with Mr 204266 were determined.
Despite the presence of five potential redox metal ions, reductive and reoxidative titrations indicate the presence of only four one‐electron‐accepting/donating species in the ligand‐free enzyme.
Participation of two copper ions in a binuclear copper site acting as a one‐electron acceptor may explain both the observed copper stoichiometry and the redox behaviour. The homology of the C‐terminal sequence of subunit II with one of the copper‐binding sites in nitrous‐oxide reductases provides possible ligands for complexing two copper ions in a binuclear center.
Cytochrome c oxidase, the terminal complex of the respiratory chain of mitochondria and many bacteria, catalyzes the electron transfer from cytochrome c to molecular oxygen, thereby reducing the ...latter to water. Thermus thermophilus HB8 (ATCC 27634) is an extremely thermophilic gram negative bacterium. It grows at temperatures between 47° and 85°C. Depending on the fermentation conditions and the 02-supply, this organism expresses two different oxidases known as the caa3-and the ba3-type. This chapter describes a new procedure for the preparation of monodispersed highly active ba3-type cytochrome c oxidase from Thermus thermophilus. The enzyme is isolated from the membrane using Triton X-100 and purified by subsequent anion-exchange chromatographies first in Triton X-100 and then in dodecyl-β-D-maltoside. The best crystals were grown in nonyl-β-D-glucoside using PEG 2000 as precipitant. However, these crystals show drastic changes in their cell constants if measured in a standard capillary and a relatively weak diffraction power in their native (as grown) state. In order to obtain a useful dataset, it was necessary to transform these crystals by dehydration.
