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The enzyme mechanism of the multicopper oxidase (MCO) SLAC from Streptomyces coelicolor was investigated by structural (XRD), spectroscopic (optical, EPR), and kinetics (stopped-flow) experiments on variants in which residue Tyr108 had been replaced by Phe or Ala through site-directed mutagenesis. Contrary to the more common three-domain MCOs, a tyrosine in the two-domain SLAC is found to participate in the enzyme mechanism by providing an electron during oxygen reduction, giving rise to the temporary appearance of a tyrosyl radical. The relatively low k(cat)/K(M) of SLAC and the involvement of Y108 in the enzyme mechanism may reflect an adaptation to a milieu in which there is an imbalance between the available reducing and oxidizing co-substrates. The purported evolutionary relationship between the two-domain MCOs and human ceruloplasmin appears to extend not only to the 3D structure and the mode of binding of the Cu's in the trinuclear center, as noted before, but also to the enzyme mechanism.

Plastocyanin is a small blue copper protein that shuttles electrons as part of the photosynthetic redox chain. Its redox behavior is changed at low pH as a result of protonation of the solvent-exposed copper-coordinating histidine. Protonation and subsequent redox inactivation could have a role in the down regulation of photosynthesis. As opposed to plastocyanin from other sources, in fern plastocyanin His90 protonation at low pH has been reported not to occur. Two possible reasons for that have been proposed: pi-pi stacking between Phe12 and His90 and lack of a hydrogen bond with the backbone oxygen of Gly36. We have produced this fern plastocyanin recombinantly and examined the properties of wild-type protein and mutants Phe12Leu, Gly36Pro, and the double mutant with NMR spectroscopy, X-ray crystallography, and cyclic voltammetry. The results demonstrate that, contrary to earlier reports, protonation of His90 in the wild-type protein does occur in solution with a pKa of 4.4 (+/-0.1). Neither the single mutants nor the double mutant exhibit a change in protonation behavior, indicating that the suggested interactions have no influence. The crystal structure at low pH of the Gly36Pro variant does not show His90 protonation, similar to what was found for the wild-type protein. The structure suggests that movement of the imidazole ring is hindered by crystal contacts. This study illustrates a significant difference between results obtained in solution by NMR and by crystallography.

Human lactoferrin (hLF) is an iron-binding glycoprotein involved in the host defence against infection and excessive inflammation. As the availability of (human milk-derived) natural hLF is limited, alternative means of production of this biopharmaceutical are extensively researched. Here we report the crystal structure of recombinant hLF (rhLF) expressed in the milk of transgenic cows at a resolution of 2.4 A. To our knowledge, the first reported structure of a recombinant protein produced in milk of transgenic livestock. Even though rhLF contains oligomannose- and hybrid-type N-linked glycans next to complex-type glycans, which are the only glycans found on natural hLF, the structures are identical within the experimental error (r.m.s. deviation of only 0.28 A for the main-chain atoms). Of the differences in polymorphic amino acids between the natural and rhLF variant used, only the side-chain of Asp561 could be modeled into the rhLF electron density map. Taken together, the results confirm the structural integrity of the rhLF variant used in this study. It also confirms the validity of the transgenic cow mammary gland as a vehicle to produce recombinant human proteins.