Inhibition of cytochrome c oxidase by nitrite and nitric ...

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Robert Hill Institute for Photosynthesis, Department of Molecular. Biology and Biotechnology, University of Sheffield, Western Bank,. Sheffield S10 2TN, U.K..
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Biochemical Society Transactions (2002) Volume 30, Part 3

11 Haem A synthesis in Bacillus subtilis: stucture and

13 Inhibition of cytochrome c oxidase by nitrite and nitric

function of CtaA M. Throne-Holst and L. Hederstedt Department of Cell and Organism Biology, Microbiology division, Lund University, Solvegatan 12, SE-223 62 Lund, Sweden

0xide:reversible and irreversible. A. Mann, P. Nicholls, M. Sharpe, C. E. Cooper Dept. Biol. Sci., Univ. Essex, Colchester, C 0 4 3SQ, U.K. Biol. Sci., Univ. Essex, Colchester, C 0 4 3SQ, U.K.

Haem A, as a prosthetic group, is uniquely found in terminal respiratory oxidases which reduce oxygen to water. Biosynthesis of haem A from protohaem IX (haem B) occurs in two steps with haem 0 as a stable intermediate. B. subtilis CtaA is required for the haem 0 to haem A synthetic step; the conversion of the methyl side chain on porphyrin ring D of haem 0 to a formyl group. CtaA is an integral membrane protein that might catalyse this entire synthetic step, which formally is a mono-oxygenation followed by a dehydrogenation. Isolated CtaA contains haem B and can also contain haem A. The haem B is thought to be a prosthetic group and haem A is probably product remaining on the enzyme. Using PhoA-fusions we have confirmed a proposed topology model of CtaA in the membrane with eight a-helical segments. A simple procedure for purification of preparative amounts of CtaA in detergent has been worked out. CtaA homologs contain four invariant His residues. The functional roles of these residues have been analysed by using mutagenesis. The results provide information on the function of CtaA. For example, some of the mutant variants are inactive in haem A synthesis but can bind haem B and haem 0, demonstrating that haem 0 is a substrate for CtaA.

Cytochrome c oxidase is ligated by neutral gases C O , H C N and NO in its reduced state and by weak acids H C N , H N 3 and H N 0 2 in its oxidized state. NO competes with oxygen for the reduced binuclear centre (a3FeCuB) and inhibits competitively with Kd approx 1nM. But NO is also oxidized in the binuclear centre to N 0 2 - , giving rise to a residual inhibition, in which apparent Km for oxygen has increased. Nitrite added externally also inhibits the oxidase reversibly and noncompetitively with oxygen, with Ki values of 5 mM at p H 7.4 and 1 mM at p H 6.4. Nitrite binding involves H+ together with N 0 2 - , as in inhibition by cyanide and azide. At p H 6.4, rapid nitrite inhibition is followed by a secondary phase, probably due to nitrite reduction to NO. Both NO and nitrite thus act as classical inhibitors, binding respectively the reduced and the oxidized states of the enzyme. Each can also give rise to the other by redox events. The resulting secondary inhibitions are less reversible than the primary ones. Either the inhibitory species are unusually tightly trapped o r a covalent modification of the a3FeCuB pocket has occurred.

12 Cobalamin (Vitamin B12 ) biosynthesis in Rhodobacter

14 Spectroscopic characterisation of the substrate binding

capsulatus. H.M.McGoldrick, E. Deery, M. Warren Queen Mary, University of London, E l 4NS.

properties of NADPH:protochlorophyllide oxidoreductase (POW Derren 1. H e .v es a n d C . Neil H u n t e r Robert Hill Institute for Photosynthesis, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, U.K.

In Pseudomonas denitrificans, cobalamin bios ynthesis is oxygen-dependent, employing a mono-oxygenase (CobG) to assist in the chemically challenging process of ring contraction. R.capsulatus, however, is able to make vitamin B12 in the absence of molecular oxygen, despite possessing a highly similar complement of biosynthetic genes. There is no CobG, but there are several open reading frames(0RFs) within the main vitamin B12 operon which have no annotated function and may therefore be involved in an alternative ‘anaerobic’ ring contraction mechanism. Two of these genes, ORF663 and ORF647, have been cloned and expressed as His-tagged recombinant proteins. Overproduction of ORF663 yielded a yellow protein of SOkDa, which gave a UV-visible spectrum typical of a flavoprotein. While overexpression of ORF647 resulted in the isolation of a brown, 15kDa protein, with a spectrum indicative of a [2Fe-2S] ferredoxin.Work is on-going to determine the precise roles these two proteins play in cobalamin biosynthesis.

0 2002 Biochemical Society

The reduction of the C17-Cl8 double bond of protochlorophyllide to form chlorophyllide is a key regulatory reaction in the chlorophyll biosynthetic pathway. The reaction is catalysed by the light-dependent enzyme NADPH:protochlorophyllide oxidoreductase (POR). The enzyme is one of only two enzymes in nature known to require light for catalysis, the other being D N A photolyase. The fluorescence of N A D P H becomes significantly enhanced upon binding to POR, enabling the apparent dissociation constant for N A D P H binding to be determined. We now describe how this technique has been applied to study putative N A D P H binding mutants. Low temperature fluorescence experiments have also been used to measure the binding of Pchlide to wild type POR. This represents the first detailed study of the substrate binding properties of the enzyme and will be important in elucidating the reaction mechanism.