Interpretation of effects of pH on rate constants for the oxidation of three ferrocytochromes c-551 with [Fe(CN)6]3- and [Co(phen)3]3+, and assignment of pKa values.
Effects of pH on second-order rate constants, k (25 degrees C), have been determined for the [Fe(CN)6]3- and [Co(phen)3]3+ oxidations of ferrocytochrome c-551 from Pseudomonas aeruginosa, Pseudomonas stutzeri, and Azotobacter vinelandii. For each oxidant similar directional trends are observed. With [Fe(CN)6]3-, rate constants over the pH 4-9.5 range first decrease, and then increase to plateau pH approximately equal to 9 k values of 0.96.10(5), 4.4.10(5) and 1.05.10(5) M-1.s-1, respectively. With [Co(phen)3]3+, rate constants increase in two separate well-defined stages from pH 2.5-9.5 to plateau pH approximately equal to 9 k values of 1.35.10(5), 3.6.10(5) and 1.37.10(5) M-1.s-1, respectively. From these trends, and consistent with previous NMR studies, protein pKa values of 7.16, 8.00 and 6.67, respectively, for the three reduced cytochromes c-551 are assigned to the buried propionic acid at position 7 on the haem ring. Since at pH greater than 6 the trends with pH for both [Fe(CN)6]3- and [Co(phen)3]3+ are in the same direction, it is concluded that this deprotonation results in a decrease in protein reduction potential. At pH less than 6, the trends with [Co(phen)3]3+ and [Fe(CN)6]3- are in opposite directions. Well defined pKa values of 3.6, 3.80 and 3.80 for P. aeruginosa, P. stutzeri and A. vinelandii, respectively, are observed with [Co(phen)3]3+ as oxidant. Upper limits only of pKa values less than 5.0, less than 4.1 and less than 4.5, respectively, are observed with [Fe(CN)6]3- as oxidant, which may or may not be the same as those observed for [Co(phen)3]3+. These latter pKa values are assigned to carboxylate residues at or near to the binding site(s). It is noted that charged residues are invariant on the front face (incorporating the exposed haem edge) of all three cytochromes c-551, and that there are only two carboxylates. One possibility is that the locality including both carboxylates defined by residues Asp-19, Lys-21, Lys-28 and Asp/Glu-29, serves as a binding site for both 3+ and 3- oxidants.
de Silva
D G
DG
Department of Chemistry, The University, Newcastle upon Tyne, U.K.
Sykes
A G
AG
D000595:Amino Acid Sequence; D001395:Azotobacter / Q000378:metabolism; D001426:Bacterial Proteins; D003035:Cobalt / Q000494:pharmacology*; D003574:Cytochrome c Group / Q000378:metabolism*; D004579:Electron Transport / Q000187:drug effects; D005295:Ferrocyanides / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009942:Organometallic Compounds / Q000494:pharmacology*; D010084:Oxidation-Reduction; D010618:Phenanthrolines / Q000494:pharmacology*; D011550:Pseudomonas aeruginosa / Q000378:metabolism
D001426:Bacterial Proteins; D003574:Cytochrome c Group; D005295:Ferrocyanides; D009942:Organometallic Compounds; D010618:Phenanthrolines; D003035:Cobalt; C046724:tris(diphenylphenanthroline)cobalt(III); C017167:cytochrome C(551); C031835:potassium ferrocyanide