Cytochrome cd1, reductive activation and kinetic analysis of a multifunctional respiratory enzyme.
Paracoccus pantotrophus cytochrome cd(1) is an enzyme of bacterial respiration, capable of using nitrite in vivo and also hydroxylamine and oxygen in vitro as electron acceptors. We present a comprehensive analysis of the steady state kinetic properties of the enzyme with each electron acceptor and three electron donors, pseudoazurin and cytochrome c(550), both physiological, and the non-physiological horse heart cytochrome c. At pH 5.8, optimal for nitrite reduction, the enzyme has a turnover number up to 121 s(-1) per d(1) heme, significantly higher than previously observed for any cytochrome cd(1). Pre-activation of the enzyme via reduction is necessary to establish full catalytic competence with any of the electron donor proteins. There is no significant kinetic distinction between the alternative physiological electron donors in any respect, providing support for the concept of pseudospecificity, in which proteins with substantially different tertiary structures can transfer electrons to the same acceptor. A low level hydroxylamine disproportionase activity that may be an intrinsic property of cytochromes c is also reported. Important implications for the enzymology of P. pantotrophus cytochrome cd(1) are discussed and proposals are made about the mechanism of reduction of nitrite, based on new observations placed in the context of recent rapid reaction studies.
Richter
Carsten D
CD
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU and School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom.
Allen
James W A
JW
Higham
Christopher W
CW
Koppenhofer
Alrik
A
Zajicek
Richard S
RS
Watmough
Nicholas J
NJ
Ferguson
Stuart J
SJ
D000818:Animals; D003574:Cytochrome c Group; D003580:Cytochromes / Q000378:metabolism*; D003575:Cytochromes c1 / Q000378:metabolism; D004579:Electron Transport; D004789:Enzyme Activation; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009572:Nitrite Reductases / Q000378:metabolism*; D010084:Oxidation-Reduction; D010230:Paracoccus / Q000201:enzymology
D003574:Cytochrome c Group; D003580:Cytochromes; C019315:cytochrome cd1; D003575:Cytochromes c1; D009572:Nitrite Reductases