Proton NMR of the histidines of azurin from Alcaligenes faecalis: linkage of histidine-35 with redox kinetics.

Abstract

On the basis of redox kinetic studies, Rosen and Pecht [Rosen, P. & Pecht, I. (1976) Biochem. 120,[339][340][341][342][343][344] observed that the azurin from the bacterium Alcaligenesfaecalis shows no such slowly attained equilibrium between two forms. Therefore, a 1H NMR study was carried out on this azurin with emphasis on the downfield region. A resonance was found at 7.95 ppm downfield that does not move with pH, is not seen in the oxidized protein, has the same pseudocontact shift in the Co(II) derivative as the C-2 proton of histidine-35 has in the Co(E) derivative of P. aeruginosa azurin, and, in the apoprotein, exhibits a typical protonation shift downfield at pH <5. Therefore, this resonance is assigned to the C-2 proton of histidine-35. The crystal structure ofP. aeruginosa azurin shows that at pH 7 the imidazole side chain ofhistidine-35 is in a crevice within the protein, where its ring is adjacent and parallel to that ofhistidine-47, a copper ligand. The preceding observations combined with others show that the kinetics of some redox reactions involving azurin depend on the position of histidine-35. The implication is that there is a pathway for electron transport to the copper atom involving passage through histidine-35.An azurin is a deep blue, low molecular weight (Mr 14,000), bacterial protein that contains one Cu(II) per molecule. The crystal structure of one of them, the azurin from Pseudomonas aeruginosa has been solved (1,2), and the copper ligands are nitrogen atoms from His-46 and His-117 and sulfur atoms from Cys-112 and Met-121. These four amino acids (and many others) are invariant in all of the azurins with sequences determined so far (3). Indeed, the deep blue color has been convincingly explained as being due to a charge-transfer transition from the cysteinate sulfur to Cu(II) (4, 5).The function of azurins in bacteria has not yet been elucidated, but a generous hint is furnished by the fast rate (6) of electron transfer between P. aeruginosa azurin and cytochrome c551, another small protein found in the same bacterium. When the temperature is suddenly raised, the redox equilibrium shifts toward reduced azurin with a fast time constant and then shifts back again with a slow time constant (7,8). Thus, there are two interesting aspects to the reaction. One is the rapid electron transfer (k 106 M -1sec 1) between two proteins, both ofwhose metal ions are well shielded from the solvent. The other is the slow interconversion of two conformers of the same protein, reduced azurin, one ofwhich is much more reactive in electron transfer than the other. Previous NMR (9-11) and temperaturejump studies (unpublished data) have shown that the slowly attained protonation-deprotonation equilibrium of the imidazole side chains of His-35 may be the slow step in the redox process. Normally the exchange time near neutral pH is short (<1 psec). The long exchange time of 0O. 1 sec actually found shows that the protonation must be accompanied by a local conformation change that alters the ef...

pH dependence of the redox reaction of azurin with cytochrome c551: role of His-35 of azurin in electron transfer

“…Az(II) at 24 °C, and 6.8 in Ale. Az(I and II) at 35 °C (Mitra & Bersohn, 1982). As previously discussed His-35 does not titrate in Ale.…”

Resolution of two distinct electron transfer sites on azurin

“…It is worth noting that the slow isomerization was not observed in the Alcaligenes faecalis azurin-Pseudomonas cy-tochrome c551 electron exchange reaction (Rosen, 1977;Wherland & Pecht, 1978;Rosen et al, 1981). It has also been shown that the homologous histidine residue (His-35; Ambler, 1971) is not involved in any acid-base equilibrium at neutral pH, most probably because the imidazole is even less accessible to the solvent in Alcaligenes as compared to Pseudomonas azurin (Mitra & Bersohn, 1982).…”

Sequential proton NMR assignments of iron(II) cytochrome c551 from Pseudomonas aeruginosa

“…The interaction between these proteins is weak (D. J. Detlefsen and Although the X-ray crystal structures of both the ferric and ferrous cytochrome c551 are known to 1.6-A resolution (Matsuura et al, 1982), there is little data available to analyze solution structural characteristics. The reduced form of azurin has at least two conformations that appear to be related to the protonation state of His 35 (Mitra & Bersohn, 1982). The oxidized form of cytochrome c551 is also conformationally labile, although it is unknown whether pH affects this conversion.…”