Effects of Engineering Uphill Electron Transfer into the Methylamine Dehydrogenase−Amicyanin−Cytochrome <i>c</i>-551i Complex

Sun, Dapeng; Davidson, Victor L.

doi:10.1021/bi0271594

Cited by 11 publications

(11 citation statements)

References 35 publications

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“…Essentially the same values of λ and H AB were obtained from each analysis. Furthermore, alteration of ΔG o by site directed mutagenesis of amicyanin caused changes in k ET which were consistent with the predictions of ET theory (33). It is likely that the large magnitude of λ is more a consequence of the TTQ cofactor of MADH than of the copper center of amicyanin (34).…”

Section: Discussionsupporting

confidence: 82%

Correlation of Rhombic Distortion of the Type 1 Copper Site of M98Q Amicyanin with Increased Electron Transfer Reorganization Energy

2007

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Mutation of the axial Met ligand of the type 1 copper site of amicyanin to Ala or Gln yielded M98A amicyanin, which exhibits typical axial type 1 ligation geometry but with a water molecule providing the axial ligand, and M98Q amicyanin, which exhibits significant rhombic distortion of the type 1 site (Carrell, C. J., Ma, J. K., Antholine, W. E., Hosler, J. P., Mathews, F. S., and Davidson, V. L. (2007) Biochemistry 46, 1900-1912). Despite the change of the axial ligand, the M98Q and M98A mutations had little effect on the redox potential of copper. The true electron transfer (ET) reactions from O-quinol methylamine dehydrogenase to oxidized native and mutant amicyanins revealed that the M98A mutation had little effect on kET, but the M98Q mutation reduced kET 45-fold. Thermodynamic analysis of the latter showed that the decrease in kET was due to an increase of 0.4 eV in the reorganization energy (lambda) associated with the ET reaction to M98Q amicyanin. No change in the experimentally determined electronic coupling or ET distance was observed, confirming that the mutation had not altered the rate-determining step for ET and that this was still a true ET reaction. The basis for the increased lambda is not the nature of the atom that provides the axial ligand because each uses an oxygen from Gln in M98Q amicyanin and from water in M98A amicyanin. Comparisons of the distance of the axial copper ligand from the equatorial plane that is formed by the other three copper ligands in isomorphous crystals of native and mutant amicyanins at atomic resolution indicate an increase in distance from 0.20 A in the native to 0.42 A in M98Q amicyanin and a slight decrease in distance for M98A amicyanin. This correlates with the rhombic distortion caused by the M98Q mutation that is clearly evident in the EPR and visible absorption spectra of the protein and suggests that the extent of rhombicity of the type 1 copper site influences the magnitude of lambda.

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Section: Discussionsupporting

confidence: 82%

Correlation of Rhombic Distortion of the Type 1 Copper Site of M98Q Amicyanin with Increased Electron Transfer Reorganization Energy

2007

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“…Previous studies of the ET reactions of native (16) and P94F (18) amicyanin with MADH and cytochrome c-551i established the validity of this ternary protein complex as a system for analysis of ET reaction rates by ET theory (eqs 4 and 5). It was demonstrated that the changes in ∆G°caused by the P94F mutation yielded predictable changes in k ET values that were consistent with true nonadiabatic ET reactions to and from the copper center.…”

Section: Resultsmentioning

confidence: 96%

“…Electron Transfer Reactions of P94A Amicyanin. The rates of the ET reactions from O-quinol MADH to oxidized P94A amicyanin and from reduced P94A amicyanin to oxidized cytochrome c-551i were determined as described previously for study of the analogous reactions of P94F amicyanin (18). An On-Line Instruments (OLIS, Bogart, GA) RSM16 stopped-flow rapid scanning spectrophotometer was used for kinetic measurements.…”

Section: Methodsmentioning

confidence: 99%

“…The increased E m values of P94F and P94A amicyanin within the ET protein complex introduce a large energy barrier for the second ET step in this three-protein ET chain. Previous studies with P94F amicyanin (18) have shown that, as a consequence of the changes in ∆E m , the k ET from TTQ to copper exhibits a 6-fold increase and the k ET from copper to heme exhibits a 100-fold decrease. These changes in k ET with ∆G°are consistent with the predictions of Marcus theory (17).…”

mentioning

confidence: 92%

“…The H AB value for ET to the cytochrome was unaffected, and the H AB for ET from MADH was slightly decreased, probably because of subtle changes in the MADH-amicyanin interface. Steady-state kinetic studies indicated that, despite the large energy barrier for the ET from copper to heme, methylamine-dependent reduction of heme by the three-protein complex with P94F amicyanin goes to completion (18).…”

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confidence: 99%

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Site-Directed Mutagenesis of Proline 94 to Alanine in Amicyanin Converts a True Electron Transfer Reaction into One That Is Kinetically Coupled

Sun

Mathews

et al. 2005

Biochemistry

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Amicyanin is a type I copper protein that mediates electron transfer (ET) from methylamine dehydrogenase (MADH) to cytochrome c-551i. Pro(94) resides in the "ligand loop" of amicyanin, a sequence of amino acids that contains three of the four copper ligands. ET from the reduced O-quinol tryptophan tryptophylquinone of MADH to oxidized P94A amicyanin is a true ET reaction that exhibits values of electronic coupling (H(AB)) and reorganization energy (lambda) that are the same as for the reaction of native amicyanin. In contrast, the parameters for the ET reaction from reduced P94A amicyanin to oxidized cytochrome c-551i have been significantly altered as a consequence of the mutation. These values of H(AB) and lambda are 8.3 cm(-)(1) and 2.3 eV, respectively, compared to values of 0.3 cm(-)(1) and 1.2 eV for the reaction of native reduced amicyanin. The crystal structure of reduced P94A amicyanin exhibits two alternate conformations with the positions of the copper 1.4 A apart [Carrell, C. J., Sun, D., Jiang, S., Davidson, V. L., and Mathews, F. S. (2004) Biochemistry 43, 9372-9380]. In one of these, conformation B, a water molecule has replaced Met(98) as a copper ligand, and the ET distance to the heme of the cytochrome is increased by 1.4 A. Analysis of these structures suggests that the true k(ET) for ET from the copper in conformation B to heme would be much less than for ET from conformation A. A novel kinetic mechanism is proposed to explain these data in which the reduction of Cu(2+) by methylamine dehydrogenase is a true ET reaction while the oxidation of Cu(1+) by cytochrome c-551i is kinetically coupled ET. By comparison of the temperature dependence of the observed rate of the coupled ET reaction from reduced P94A amicyanin to cytochrome c-551i with the predicted rates and temperature dependence for the true ET reaction from conformation A, it was possible to determine the K(eq) and values of DeltaH degrees and DeltaS degrees that are associated with the non-ET reaction that modulates the observed ET rate.

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Electron transfer in crystals of the binary and ternary complexes of methylamine dehydrogenase with amicyanin and cytochrome c551i as detected by EPR spectroscopy

et al. 2004

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EPR studies of the methylamine dehydrogenase (MADH)-amicyanin and MADH-amicyanin-cytochrome c551i crystalline complexes have been performed on randomly oriented microcrystals before and after exposure to the substrate, methylamine, as a function of pH. The results show that EPR signals from the redox centers present in the various proteins can be observed simultaneously. These results complement and extend earlier studies of the complexes under similar conditions that utilized single-crystal polarized absorption microspectrophotometry. The binary complex shows a blue copper axial signal, characteristic of oxidized amicyanin. After reaction of substrate with the MADH coenzyme tryptophan tryptophylquinone (TTQ), the binary complex exhibits an equilibrium mixture of oxidized copper/reduced TTQ and reduced copper/TTQ. radical, whose ratio is dependent on the pH. In the oxidized ternary complex, the same copper axial signal is observed superimposed on the low-spin ferric heme features characteristic of oxidized cytochrome c551i. After addition of substrate to the ternary complex, a decrease of the copper signal is observed, concomitant with the appearance of the radical signal derived from the semiquinone form of TTQ. The equilibrium distribution of electrons between TTQ and copper as a function of pH is similar to that observed for the binary complex. This result was essential to establish that the copper center retains its function within the crystalline ternary complex. At high pH, with time the low-spin heme EPR features disappear and the spectrum indicates that full reduction of the complex by substrate has occurred.

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Effects of Engineering Uphill Electron Transfer into the Methylamine Dehydrogenase−Amicyanin−Cytochrome c-551i Complex

Cited by 11 publications

References 35 publications

Correlation of Rhombic Distortion of the Type 1 Copper Site of M98Q Amicyanin with Increased Electron Transfer Reorganization Energy

Correlation of Rhombic Distortion of the Type 1 Copper Site of M98Q Amicyanin with Increased Electron Transfer Reorganization Energy

Site-Directed Mutagenesis of Proline 94 to Alanine in Amicyanin Converts a True Electron Transfer Reaction into One That Is Kinetically Coupled

Electron transfer in crystals of the binary and ternary complexes of methylamine dehydrogenase with amicyanin and cytochrome c551i as detected by EPR spectroscopy

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