Ca²⁺-Binding Site in Rhodobacter Sphaeroides Cytochrome c Oxidase

Abstract

Cytochrome c oxidase (COX) from R. sphaeroides contains one Ca(2+) ion per enzyme that is not removed by dialysis versus EGTA. This is similar to COX from Paracoccus denitrificans [Pfitzner, U., Kirichenko, A., Konstantinov, A. A., Mertens, M., Wittershagen, A., Kolbesen, B. O., Steffens, G. C. M., Harrenga, A., Michel, H., and Ludwig, B. (1999) FEBS Lett. 456, 365-369] and is in contrast to the bovine oxidase, which binds Ca(2+) reversibly. A series of R. sphaeroides mutants with replacements of the E54, Q61, and D485 residues, which form the Ca(2+) coordination sphere in subunit I, has been generated. The substitutions for the E54 residue do not assemble normally. Mutants with the Q61 replacements are active and retain the tightly bound Ca(2+); their spectra are not perturbed by added Ca(2+) or EGTA. The D485A mutant is active, binds to Ca(2+) reversibly, like the mitochondrial oxidase, and exhibits the red shift in the heme a absorption spectrum upon Ca(2+) binding for both reduced and oxidized states of heme a. The K(d) value of 6 nM determined by equilibrium titrations is much lower than that reported for the homologous D477A mutant of Paracoccus denitrificans or for bovine COX (K(d) = 1-3 microM). The rate of Ca(2+) binding with the D485A oxidase (k(on) = 5 x 10(3) M(-1) s(-1)) is comparable to that observed earlier for bovine COX, but the off-rate is extremely slow (approximately 10(-3) s(-1)) and highly temperature-dependent. The k(off) /k(on) ratio (190 nM) is about 30-fold higher than the equilibrium K(d) of 6 nM, indicating that formation of the Ca(2+)-adduct may involve more than one step. Sodium ions reverse the Ca(2+)-induced red shift of heme a and dramatically decrease the rate of Ca(2+) binding to the D485A mutant COX. With the D485A mutant, 1 Ca(2+) competes with 1 Na(+) for the binding site, whereas 2 Na(+) compete with 1 Ca(2+) for binding to the bovine oxidase. This finding indicates that the aspartic residue D442 (a homologue of R. sphaeroides D485) may be the second Na(+) binding site in bovine COX. No effect of Ca(2+) binding to the D485A mutant is evident on either the steady-state enzymatic activity or several time-resolved partial steps of the catalytic cycle. It is proposed that the tightly bound Ca(2+) plays a structural role in the bacterial oxidases while the reversible binding with the mammalian enzyme may be involved in the regulation of mitochondrial function.

Functional Importance of a Pair of Conserved Glutamic Acid Residues and of Ca2+ Binding in the cbb3-Type Oxygen Reductases from Rhodobacter sphaeroides and Vibrio cholerae

“…The role of the Ca 2+ to form a bridge between the CcoN and CcoO subunits is also critical, as evidenced by the lack of protein assembly when the Ca 2+ -liganding amino acid in the CcoO subunit (S105) is substituted by another amino acid. The essential nature of the Ca 2+ in the cbb 3 oxygen reductases is in contrast to the Ca 2+ which binds near the low-spin heme in some of the A-family oxygen reductases, but which is not required for assembly or enzyme activity (25, 27). …”

Primary Metabolism of Chickpea Is the Initial Target of Wound Inducing Early Sensed Fusarium oxysporum f. sp. ciceri Race I

“…The octadecanoid pathway along with the lipoxygenase pathway is responsible for the production of oxylipins that are important for plant defense [76]. Cytochrome C oxidase (COX), the key enzyme of aerobic respiration, is involved in the translocation of protons and has an active role in regulating stress-mediated signals [77]. The basal expression of the ribosomal protein transcripts and oxidase enzymes related ESTs in the resistant variety emphasizes their role in transcriptional regulation and signal generation probably due to pathogen-induced enhanced respiration throughout the cell during early infection.…”

Seven perspectives on GPCR H/D-exchange proteomics methods

“…Previous projects used K + -based buffers to purify physiological-state high-activity C c O because both mitochondria and Rs bacteria have high internal K + , whereas Na + was empirically screened as a tool to aid crystallogenesis 29, 42– 44 . Na + and K + affect C c O Ca 2+ binding differently 45, 46 , though the exact actions remain obscure. For GPCRs, unique among all common cations, Na + was long-observed to act as a fast allosteric mediator itself and control agonist/antagonist-distinct GPCR activities, and the structural bases started to be resolved by crystallography 26, 36, 47– 56 .…”