Limei Hsu Jones

Intermolecular electron transfer from quinoproteins and its relevance to biosensor technology

Abstract Long-range intermolecular electron transfer occurs between the redox centers of the quinoprotein methylamine dehydrogenase and the copper protein amicyanin. A steady-state kinetic assay was developed and used to describe factors that are relevant to this process. Kinetic, physical, and structural data on these proteins and the complexes which they form are providing a picture of how nature accomplishes long-range electron transfer through proteins, a process that is critical to the development of direct amperometric biosensors.

Binding constants for a physiologic electron-transfer protein complex between methylamine dehydrogenase and amicyanin. Effects of ionic strength and bound copper on binding.

Two soluble proteins, methylamine dehydrogenase and amicyanin, form a physiologically relevant complex in which intermolecular electron transfer occurs. To characterize and quantitate the binding of these two weakly-associating proteins, an ultrafiltration binding assay has been developed which involves brief centrifugation of mixtures of proteins in centrifuge concentrators followed by quantitation of proteins on each side of the filtration membrane by HPLC. Under low ionic strength conditions which are optimal for the redox reaction between these proteins, a Kd of 4.5 microM was measured for the methylamine dehydrogenase-amicyanin complex. The Kd increased by 8-fold in the presence of added salt. Apoamicyanin, which is known from crystallographic analysis to be structurally very similar to amicyanin, exhibited a much higher Kd and much less specific binding than did the holoprotein. Apoamicyanin also exhibited apparent self-association at low ionic strength which was not observed with amicyanin. These observations are correlated with the known crystal structures of these proteins, free and in complex, and with the available biochemical information on the interactions of these two proteins.

Complex Formation with Methylamine Dehydrogenase Affects the Pathway of Electron Transfer from Amicyanin to Cytochrome c-551i

Methylamine dehydrogenase (MADH), amicyanin, and cytochrome c-551i are soluble redox proteins that form a complex in solution (Chen, L., Durley, R., Mathews, F. S., and Davidson, V. L.(1994) Science 264, 86-90), which is required for the physiologic electron transfer from the tryptophan tryptophylquinone cofactor of MADH to heme via the copper center of amicyanin. The reduction of cytochrome by amicyanin within the complex in solution has been demonstrated using rapid scanning stopped-flow spectroscopy. Electron transfer from free, uncomplexed, amicyanin to cytochrome c-551i occurs much more rapidly but only to a very small extent because the reaction is thermodynamically much less favorable when amicyanin is not associated with MADH (Gray, K. A., Davidson, V. L., and Knaff, D. B.(1988) J. Biol. Chem. 263, 13987-13990). These kinetic data suggest that amicyanin binding to cytochrome c-551i occurs at different sites when amicyanin is free and when it is in complex with MADH. A model for the interactions of these proteins is presented.

Cofactor-directed inactivation by nucleophilic amines of the quinoprotein methylamine dehydrogenase from Paracoccus denitrificans

Phenylhydrazine, semicarbazide, aminoguanidine, hydrazine, and hydroxylamine each irreversibly inactivated methylamine dehydrogenase from Paracoccus denitrificans and caused changes in the absorbance spectrum of the protein-bound tryptophan tryptophylquinone [TTQ] prosthetic group. Different spectral perturbations were observed on reaction with each of these inactivators. In each case a stoichiometry of 2 mol per mol of enzyme (1:1 per cofactor) was required to observe complete modification of the absorbance spectrum. Identical changes were observed in the presence and absence of oxygen. The reactions of hydrazine and hydroxylamine were very rapid, with stoichiometric inactivation occurring in less than 30 s. Inactivation by phenylhydrazine and semicarbazide exhibited apparent bimolecular kinetics and second order rate constants for inactivation, respectively, of 25 min-1 mM-1 and 39 min-1 mM-1. In contrast, inactivation by aminoguanidine exhibited saturation behavior and kinetic parameters of KI = 2.5 mM and kinact = 0.5 min-1 were obtained. Ammonium salts did not inactivate the enzyme, but were reversible competitive inhibitors with respect to methylamine. A Ki of 20 mM was obtained for ammonium chloride. A mechanism for the reactions of these compounds with the TTQ cofactor of methylamine dehydrogenase is proposed, and the relationship of these data to the mechanisms of interaction of these compounds with o-quinones and other quinoproteins which possess TTQ and other quinone cofactors is discussed.

Active Site Aspartate Residues Are Critical for Tryptophan Tryptophylquinone Biogenesis in Methylamine Dehydrogenase

The biosynthesis of methylamine dehydrogenase (MADH) requires formation of six intrasubunit disulfide bonds, incorporation of two oxygens into residue βTrp57 and covalent cross-linking of βTrp57 to βTrp108 to form the protein-derived cofactor tryptophan tryptophylquinone (TTQ). Residues βAsp76 and βAsp32 are located in close proximity to the quinone oxygens of TTQ in the enzyme active site. These residues are structurally conserved in quinohemoprotein amine dehydrogenase, which possesses a cysteine tryptophylquinone cofactor. Relatively conservative βD76N and βD32N mutations resulted in very low levels of MADH expression. Analysis of the isolated proteins by mass spectrometry revealed that each mutation affected TTQ biogenesis. βD76N MADH possessed the six disulfides but had no oxygen incorporated into βTrp57 and was completely inactive. The βD32N MADH preparation contained a major species with six disulfides but no oxygen incorporated into βTrp57 and a minor species with both oxygens incorporated, which was active. The steady-state kinetic parameters for the βD32N mutant were significantly altered by the mutation and exhibited a 1000-fold increase in the Km value for methylamine. These results have allowed us to more clearly define the sequence of events that lead to TTQ biogenesis and to define novel roles for aspartate residues in the biogenesis of a protein-derived cofactor.

Tyr30 of amicyanin is not critical for electron transfer to cytochrome c-551i: implications for predicting electron transfer pathways

A Pathways analysis of the methylamine dehydrogenase-amicyanin-cytochrome c-551i protein electron transfer (ET) complex predicts two sets of ET pathways of comparable efficiency from the type I copper of amicyanin to the heme of cytochrome c-551i. In one pathway, the electron exits copper via the Cys(92) copper ligand, and in the other, it exits via the Met(98) copper ligand. If the Pathways algorithm is modified to include contributions from the anisotropy of metal-ligand coupling, independent of differences in copper-ligand bond length, then the pathways via Cys(92) are predicted to be at least 100-fold more strongly coupled than the pathways via any of the other copper ligands. All of the favored pathways via Cys(92) include a through-space jump from Cys(92) to the side chain of Tyr(30). To determine whether or not the pathways via Cys(92) are preferentially used for ET, Tyr(30) was changed to other amino acid residues by site-directed mutagenesis. Some mutant proteins were very unstable suggesting a role for Tyr(30) in stabilizing the protein structure. Y30F and Y30I mutant amicyanins could be isolated and analyzed. For the Y30I mutant, the modified Pathways analysis which favors ET via Cys(92) predicts a decrease in ET rate of at least two orders of magnitude, whereas the standard Pathways analysis predicts no change in ET rate since ET via Met(98) is not affected. Experimentally, the ET rates of the Y30I and Y30F mutants were indistinguishable from that of wild-type amicyanin. Likely explanations for these observations are discussed as are their implications for predicting pathways for ET reactions of metalloproteins.

Reaction mechanism for the inactivation of the quinoprotein methylamine dehydrogenase by phenylhydrazine

Phenylhydrazine has previously been shown to be an irreversible inactivator of the tryptophan tryptophylquinone (TTQ) enzyme methylamine dehydrogenase [Davidson, V.L. and Jones, L.H. (1992) Biochim. Biophys. Acta 1121, 104-110]. Structure-reactivity correlations have been performed to elucidate the mechanism by which this inactivation occurs. The reactions of a series of p-substituted phenylhydrazines with methylamine dehydrogenase were examined. Correlation with electronic substituent effects was observed. A Hammett plot of the second order inactivation rate constants versus sigma p exhibited a positive slope. Plots of these rate constants against substituent constants which reflected either resonance or field/inductive parameters for each p-substituent indicated that the rate was primarily influenced by resonance electronic effects. A Brønsted plot of the inactivation rate constant against pKa of each substituted phenylhydrazine yielded a beta-value (slope) of 0.7. Based upon these results, a reaction mechanism is proposed for the inactivation of methylamine dehydrogenase by phenylhydrazines, and a structure is proposed for the putative transition state for the rate-limiting step in the overall processes of binding and adduct formation by phenylhydrazine. The relevance of these results to the process of imine formation between substrate amines and TTQ during the normal catalytic process is also discussed.