Six- to Five-Coordinate Heme-Nitrosyl Conversion in Cytochrome c′ and Its Relevance to Guanylate Cyclase † Colin R. Andrew,* ,‡,§ Simon J. George, | David M. Lawson, | and Robert R. Eady | Department of Chemistry, Eastern Oregon UniVersity, One UniVersity BouleVard, La Grande, Oregon 97850-2899, Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, 20000 NW Walker Road, BeaVerton, Oregon 97006-8921, and Department of Biological Chemistry, John Innes Centre, Norwich NR4 7UH, U.K. ReceiVed July 9, 2001 ABSTRACT: The 5-coordinate ferrous heme of Alcaligenes xylosoxidans cytochrome c′ reacts with NO to form a 6-coordinate nitrosyl intermediate (λ Soret at 415 nm) which subsequently converts to a 5-coordinate nitrosyl end product (λ Soret at 395 nm) in a rate-determining step. Stopped-flow measurements at pH 8.9, 25 °C, yield a rate constant for the formation of the 6-coordinate nitrosyl adduct, k on ) (4.4 ( 0.5) × 10 4 M -1 s -1 , which is 3-4 orders of magnitude lower than the values for other pentacoordinate ferrous hemes and is consistent with NO binding within the sterically crowded distal heme pocket. Resonance Raman measurements of the freeze-trapped 6-coordinate nitrosyl intermediate reveal an unusually high Fe-NO stretching frequency of 579 cm -1 , suggesting a distorted Fe-N-O coordination geometry. The rate of 6- to 5-coordinate heme nitrosyl conversion is also dependent upon NO concentration, with a rate constant, k 6-5 ) (8.1 ( 0.7) × 10 3 M -1 s -1 , implying that an additional molecule of NO is required to form the 5c-NO adduct. Since crystallographic studies have shown that the 5-coordinate nitrosyl complex of cytochrome c′ binds NO to the proximal (rather than distal) face of the heme, the NO dependence of the 6- to 5-coordinate NO conversion supports a mechanism in which the weakened His ligand, as well as the distally bound NO, is displaced by a second NO molecule which attacks and is retained in the proximal coordination position. The fact that a dependent 6- to 5-coordinate nitrosyl conversion has been previously reported for soluble guanylate cyclase suggests that the mechanism of Fe-His bond cleavage may be similar to that of cytochrome c′ and strengthens the recent proposal that both proteins exhibit proximal NO binding in their 5-coordinate nitrosyl adducts. Nitric oxide (NO) 1 is implicated as a signaling molecule in a wide range of organisms including animals, plants, and micoorganisms (1-7). The best characterized system is that of animals, in which cell-cell signaling occurs through the interaction of micromolar NO levels with the heme-contain- ing enzyme, soluble guanylate cyclase (sGC) (8). Binding of NO to the heme center of sGC triggers the conversion of GTP to the second-messenger cGMP, which in turn regulates a host of physiological processes such as smooth muscle contraction, blood clotting, and neurotransmission (2, 8). Formation of a 5c-NO heme complex in sGC and the associated Fe-His bond cleavage are believed to be the trigger which activates the production of cGMP (8, 9). Deducing the mechanism of 5c-NO adduct formation in sGC is, therefore, of particular biomedical interest. Cytochrome c′ (cyt c′) is a hemoprotein found in the periplasm of certain proteobacteria which contains a penta- coordinate heme center located toward the C-terminus of a four R-helix bundle (10, 11). Although the exact physiologi- cal role of cyt c′ is unclear, several studies have suggested that NO binding to the heme may help bacteria suppress potentially toxic levels of free NO in their environment (12- 16). In particular, Moir and co-workers have shown that a cyt c′-deficient mutant of the photosynthetic bacterium Rhodobacter capsulatus exhibited increased sensitivity to nitrosative stress (16). Intriguingly, the same group recently reported in vivo studies suggesting that R. capsulatus cyt c′ might in fact function as an NO reductase (17). Additional interest in the coordination chemistry of cyt c′ stems from similarities with sGC, including the ability to form a 5c-NO heme adduct (18). Moreover, recent crystal- lographic characterization of cyt c′ from Alcalignes xylosoxi- dans (AXCP) has yielded the exciting discovery that exogenous ligands are able to bind to the Fe from either side of the heme face. Whereas the 6c-CO complex of AXCP contains CO bound to Fe at the distal position, the 5c-NO adduct exhibits a novel proximal coordination geometry with NO residing at the site originally occupied by the His ligand (19). In the case of sGC, the currently accepted model for † This work was supported by NIH Grant GM 34468 to Professor Thomas M. Loehr. D.M.L., S.J.G., and R.R.E. are funded by the BBSRC as part of the competitive strategic grant to the John Innes Centre. * To whom correspondence should be addressed. Fax: (541) 962- 3873. E-mail: candrew@eou.edu. ‡ Present address: Eastern Oregon Univeristy. § Oregon Graduate Institute of Science and Technology. | John Innes Centre. 1 Abbreviations: NO, nitric oxide; CO, carbon monoxide; 5c and 6c, 5-coordinate and 6-coordinate, respectively; cyt c′, cytochrome c′; AXCP, Alcaligenes xylosoxidans cyt c′; cyt c, mitochondrial cytochrome c; sGC, soluble guanylate cyclase; Mb, myoglobin; Hb, hemoglobin; CCP, cytochrome c peroxidase; CooA, CO oxidation activator; PGHS- 1, prostaglandin endoperoxide H synthase-1; NOS, nitric oxide synthase; P450, cytochrome P450; CPO, chloroperoxidase. 2353 Biochemistry 2002, 41, 2353-2360 10.1021/bi011419k CCC: $22.00 © 2002 American Chemical Society Published on Web 01/25/2002