Reactive sites and course of reduction in the Rieske protein

Abstract

Rieske proteins play an essential role in electron transfer in the bc complex. Rieske proteins contain a [2Fe-2S] cluster with one iron ligated by two histidines and the other iron ligated by two cysteines. All Rieske proteins have pH-dependent reduction potentials with the histidines ligating the cluster deprotonating in response to increases in pH. The addition of diethylpyrocarbonate (DEPC) modifies deprotonated histidines. The previous studies on the isolated Thermus thermophilus Rieske protein haveused large excesses of DEPC, and this study examines what amino acids become modified under different molar equivalents of DEPC to protein. Increasing amounts of DEPC result in more modification, and higher pH values result in faster reaction. Upon modification, the protein also becomes reduced and ~6 equivalents of DEPC are needed for 50% of the reduction to occur. Which amino acids are modified first also points to the most reactive species on the protein. Mass spectrometry analysis shows that lysine 68 is the most reactive amino acid, followed by the ligating histidine 154 and two other surfaces lysines, 76 and 43. The modification of the ligating histidine at low numbers of DEPC equivalents and correlation with a similar number of equivalents needed to reduce the protein shows that this histidine can interact with neighboring groups, and these results can be extended to the protein within the bc complex, where interaction with neighboring residues or molecules may allow reduction to occur. These results may shed light on how Rieske transfers electrons and protons in the bc complex.

Charge Regulation in a Rieske Proton Pump Pinpoints Zero, One, and Two Proton-Coupled Electron Transfer

“…Recombinant truncTtRp was expressed in E. coli and purified as previously described. 18 E. coli was grown in TB containing 100 μg/mL ampicillin to an OD 600 of ∼1.0 AU. The cells were then induced with 0.4 mM IPTG, supplemented with 150 μM cysteine and 150 μg/mL ferric ammonium citrate, shaken for 18−20 h, and then pelleted via centrifugation at 5000 × g for 5 min at 4 °C.…”

“…16,17 The current study measured ΔZ ET in a truncated form of the Rieske protein from Thermus thermophilus (denoted truncTtRp). 18 The truncTtRp protein lacks 8 N-terminal and 9 C-terminal residues. A majority of the 17 truncated residues are hydrophobic and nonionizable (except for two E, one Y, and two R).…”

“…22,23 Pioneering studies of truncTtRp suggest that H154 and H134 are sequentially deprotonated at increasing pH (to imidazolate) in Fe(III) but become reprotonated upon reduction to Fe(II). 18,24 The pK a of these two histidine residues have been approximated with NMR, 2,25,26 Raman spectroscopy, 27−30 UV−vis and CD spectroscopy, 31,32 along with cyclic voltammetry: 24,33 5, it is hypothesized that two protons are coupled to electron transfer (i.e., H154 and H134 are both protonated upon reduction). We refer to this latter process as "2PCET."…”

DEPC modification of the CuA protein from Thermus thermophilus

“…It is not unprecedented that a ligating histidine is one of the residues most susceptible to DEPC modification. Analysis of the Thermus thermophilus Rieske protein using mass spectrometry showed that the ligating histidine His154 is one of the first amino acids modified by DEPC [55]. Together these cases indicate that ligating histidines can be extremely reactive toward DEPC, comparable even to non-ligating histidines and lysines.…”

“…There is significant spectroscopic evidence for modification of a ligating histidine, but modification of tyrosines and lysines may contribute to the altered CD spectra of TtCu A and H40A/H117A through structural alterations of the proteins. As primary amines, lysines are often the most reactive residues toward DEPC [55]; however, all lysines in TtCu A are located approximately 20 Å or further from the metal center, making lysine modification highly unlikely to influence metal center properties except through large-scale conformational changes. Large conformational changes are not observed as the far UV-CD spectrum of TtCu A only shifts slightly after the addition of DEPC (data not shown),…”