Functional and Structural Roles of Residues in the Third Extramembrane Segment of Adrenal Cytochrome b₅₆₁

Abstract

Several residues in the third extramembrane segment (EM3) of adrenal cytochrome b561 have been proposed to be involved in this cytochrome’s interaction with ascorbate, but there has been no systematic evaluation of residues in the segment. We used alanine-scanning mutagenesis to assess the functional and structural roles of the EM3 residues and several adjacent residues (residues 70–85) in the bovine cytochrome. Each alanine mutant was expressed in a bacterial system, detergent solubilized, and affinity purified. The recombinant proteins contained ~two hemes/monomer and, except for R74A, retained basic functionality (≥ 94% reduced by 20 mM ascorbate). Equilibrium spectrophotometric titrations with ascorbate were used to analyze the alpha band lineshape and amplitude during reduction of the high- and low-potential heme centers (bH and bL, respectively), and the midpoint ascorbate concentrations for the bH and bL transitions (CH and CL, respectively). Y73A and K85A markedly narrowed the bH alpha band peak; other mutants had lesser or no effects on bH or bL spectra. Relative changes in CH for the mutants were larger than changes in CL, with increases of 1.5- to 2.9-fold in CH for L70A, L71A, Y73A, R74A, N78A and K85A. The amounts of functional bH and bL centers in additional Arg74 mutants, assessed by ascorbate titration and EPR spectroscopy, declined in concert in the order: wildtype > R74K > R74Q > R74T, R74Y > R74E. The results of this first comprehensive experimental test of the proposed roles of EM3 residues have identified residues with direct or indirect impact on ascorbate interactions, on the environment of the bH heme center, and on formation of the native bH/bL unit. Surprisingly, no individual EM3 residue was by itself indispensable for the interaction with ascorbate, and the role of the segment appears to be more subtle than previously thought. The present results also support our topological model of the adrenal cytochrome, which positions bH near the cytoplasmic side of the membrane.

Proton-Coupled Electron Transport in Two Distinct CYBASC Paralogs of Arabidopsis thaliana: A Comparative Characterization of Highly Conserved Tyrosine and Lysine Residues

“…Furthermore, and also in contrast to our results for both paralogs, the electron acceptance of Zm -Y71F was accelerated . A comparable change in the α-band shape as seen for A-Y69F in this study has been shown for the corresponding Bt Cyt b 561 -CG variant Y73A as well as for the variant K85A (corresponds to A-K80), whereupon the authors suggested a cation−π-type interaction , between those amino acids based on a bioinformatic model. , …”

“…14 A comparable change in the α-band shape as seen for A-Y69F in this study has been shown for the corresponding BtCytb 561 -CG variant Y73A as well as for the variant K85A (corresponds to A-K80), 45 whereupon the authors suggested a cation−π-type interaction 46,47 between those amino acids based on a bioinformatic model. 38,45 BtCytb 561 -CG exhibits 31.7% and 33.8% sequence identity with the A-and B-paralogs, respectively, and Zmcytb561 exhibits values of 63.5% and 39.7%, respectively. Should a proton-coupled electron transfer (PCET) be active in CYBASC proteins, as proposed for Btcytb 561 -CG 16,48 and Zmcytb 561 , 5,49 the conserved tyrosine residue is possibly involved in this process by proton transfer to one of the heme b-coordinating histidine residues.…”

“…Substitution of lysine with glutamate (A-K76E) resulted in a moderate decrease in the RCA to ∼43%, which is still in agreement with its proposed function in substrate stabilization. The distinct upward shift of E M ( b H ) in A-K76E of 75 mV might also be explained by the presence of a negative instead of a positive charge, because E M values of heme redox centers are also modulated by the interaction of charges with their propionate side chains. − ,, − Furthermore, the impact on only E M ( b H ) clearly demonstrates the localization of b H on the site of AscH – oxidation in the A-paralog as proposed previously. ,, Surprisingly, mutation of K76 to isoleucine (A-K76I) accelerated the AscH – oxidation kinetics to an RCA of almost 150% (Table ), whereas both E M values were found to be in the range of those of the wild type (Table and Figure S5). We conclude that the function of lysine 76 in the A-paralog is only partially comparable to that of B-K77 and exhibits subtle differences, possibly due to a different relative orientation with respect to other basic amino acids, the conserved A-Y69, and functional groups of b H .…”

“…18−21,41,63−65 Furthermore, the impact on only E M (b H ) clearly demonstrates the localization of b H on the site of AscH − oxidation in the Aparalog as proposed previously. 3,45,66 Surprisingly, mutation of K76 to isoleucine (A-K76I) accelerated the AscH − oxidation kinetics to an RCA of almost 150% (Table 1), whereas both E M values were found to be in the range of those of the wild type (Table 1 and Figure S5). We conclude that the function of lysine 76 in the A-paralog is only partially comparable to that of B-K77 and exhibits subtle differences, possibly due to a different relative orientation with respect to other basic amino acids, the conserved A-Y69, and functional groups of b H .…”

Functional Assembly of Caenorhabditis elegans Cytochrome b-2 (Cecytb-2) into Phospholipid Bilayer Nanodisc with Enhanced Iron Reductase Activity

“…They have a unique structure including six hydrophobic transmembrane α-helices being bound with two heme b prosthetic groups [ 1 , 2 ]. The redox potential measurements for the purified proteins indicated that the cytosolic ascorbate is the electron donor for the CYB561 proteins [ 3 , 4 ]. Furthermore, some members of CYB561 showed a distinct ferric reductase activity that could have a vital role for the iron metabolism of eukaryotic cells [ 5 ].…”